Compositions and methods for making mutations in cell lines and animals

ABSTRACT

Abstract of Disclosure 
     The present invention is directed generally to reduction or inactivation of gene function or gene expression in cells in vitro and in multiorganisms.  The invention encompasses methods for mutating cells using a combination of mutagens, particularly wherein at least one mutagen is an insertional mutagen, to achieve homozygous gene mutation or mutation of multiple genes required cumulatively to achieve a phenotype to create  knock-out s,  knock-down s, and other modifications in the same cell.  The invention is also directed to cells (and libraries thereof) and organisms created by the methods of the invention, including those in which at least one of the genes created by insertional mutagenesis is tagged by means of the insertion sequences thereby allowing identification of the mutated gene(s).  The invention is also directed to libraries of mutated cells and their uses.  The invention is also directed to methods of identifying mutations with methods of the invention, in cells (and libraries thereof) and organisms, by means of the insertional tag.

Cross Reference to Related Applications

[0001] The present application claims the benefit of U.S. ProvisionalApplication No. 60/336,497 filed October 22, 2001, U.S. Application No.10/196,721 filed July 15, 2002, and U.S. Patent Application Serial No.10/277,612 filed October 22, 2002, the entire disclosures of which areincorporated herein by reference.

Background of Invention

[0002]Field of the InventionThe present invention is in the fields ofmolecular biology, cell biology, and genetics. The invention is directedgenerally to mutating genes in cells in vitro and in multi-cellularorganisms. The invention encompasses methods for mutating genes in cellsusing a combination of mutagens, wherein at least one mutagen is apolynucleotide that acts as an insertional mutagen. Such methods areused to achieve mutation of a single gene to achieve a desired phenotypeas well as mutation of multiple genes, required cumulatively to achievea desired phenotype, in a cell or multi-cellular organism. The inventionis also directed to methods of identifying one or more mutated genes,made by the methods of the invention, in cells and in multi-cellularorganisms, by means of a tagging property provided by the insertionalmutagen(s). The insertional mutagen thus allows identification of one ormore genes that are mutated by insertion of the insertional mutagen.

[0003] The invention is also directed to cells and multi-cellularorganisms created by the methods of the invention and uses of the cellsand multicellular organisms. The invention is also directed to librariesof cells created by the methods of the invention and uses of thelibraries.

[0004]BackgroundMutagenesis has been used to identify the function of alarge and growing number of genes. Mutation of one or more genes in amulti-cellular organism or cell allows the artisan to study the mutantorganism or cell and compare it to the non-mutagenized (which may bewildtype) parent organism or cell. By identifying phenotypes associatedwith the mutant organism or cell, the function of the mutated gene(s)can be ascertained. Furthermore, mutagenesis provides a means foraltering the genetic make up of a cell or multi-cellular organism toobtain a desired result. For example, it may be desirable to create aphysiological disorder in a eukaryotic organism by mutating one moregenes and then to identify one or more of the relevant genes. Thus,mutations that have a desired use (e.g., for commercial production ofproteins, foodstuffs, or pharmaceuticals, or for production oftransgenic animals as models of certain diseases) can be identified andselected. The possibilities for use of this technology, whether invitro, ex vivo, or in vivo, are well known in the art.

[0005] Identification of novel genes and characterization of theirfunction using mutagenesis has also been shown to be productive inidentifying new drugs and drug targets. Creating in vitro cellularmodels that exhibit phenotypes that are clinically relevant provides avaluable substrate for target identification and screening for compoundsthat modulate not only the phenotype but also the target(s) thatcontrols the phenotype. Modulation of such a target can provideinformation that validates the target as important for therapeuticintervention in a clinical disorder when such modulation of the targetserves to modulate a clinically relevant phenotype.

[0006] Animal models exhibiting clinically relevant phenotypes are alsovaluable for drug discovery and development and for drug targetidentification. For example, mutation of somatic or germ cellsfacilitates the production of genetically modified offspring or clonedanimals having a phenotype of interest. Such animals have a number ofuses, for example as models of physiological disorders (e.g., of humangenetic diseases) that are useful for screening the efficacy ofcandidate therapeutic compounds or compositions for treating orpreventing such physiological disorders. Furthermore, identifying thegene(s) responsible for the phenotype provides potential drug targetsfor modulating the phenotype and, when the phenotype is clinicallyrelevant, for therapeutic intervention. In addition, the manipulation ofthe genetic makeup of organisms and the identification of new genes haveimportant uses in agriculture, for example in the development of newstrains of animals and plants having higher nutritional value orincreased resistance to environmental stresses (such as heat, drought,or pests) relative to their wildtype or non-mutant counterparts.

[0007] Since most eukaryotic cells are diploid, two copies of most genesare present in each cell. As a consequence, homozygous mutation isusually required to produce a desired phenotype, since mutating one copyof a gene may not produce a sufficient change in the level of geneexpression or activity of the gene product from that in the non-mutatedor wildtype cell or multicellular organism, and since the remainingwildtype copy would still be expressed at sufficient levels to produce afunctional gene product. Thus, to create a desired change in the levelof gene expression and/or function in a cell or multicellular organism,at least two mutations, one in each copy of the gene, are required inthe same cell.

[0008] In other instances, mutation in multiple genes may be required toproduce a desired phenotype. In some instances, a mutation in one copyof a gene may affect the expression levels of the gene but not theactivity of the gene product to a desired extent, so that the desiredphysiological effects on the cell or multi-cellular organism is notachieved. However, a mutation in a second gene, even in only one copy ofthat second gene, can reduce gene expression levels of the second geneto produce a cumulative phenotypic effect in combination with the firstmutation, if the expression levels of both genes are sufficiently low.This effect can alter the function of a cell or multi-cellular organism.An example of this phenomenon is the synergy between blood clottingFactors VIII and IX. A mutation in either gene alone could result inlevels that are severely reduced but with no effect on the clottingfunction. Severe reductions in the level of expression of both genes,however, can have a major impact. This principle can be extended toother instances where mutations in multiple (two, three, four, or more,for example) genes are required cumulatively to produce an effect onactivity of a gene product or on another phenotype in a cell ormulti-cellular organism. It should be noted that, in this instance, suchgenes may all be expressed in the same cell type and therefore, all ofthe required mutations occur in the same cell. However, the genes maynormally be expressed in different cell types (for example, secretingthe different gene products from the different cells). In this case, thegene products are expressed in different cells but still have abiochemical relationship such that one or more mutations in each gene isrequired to produce the desired phenotype.

[0009] Unfortunately, few methods exist for creating cultured cells thatcontain multiple gene mutations that produce, cumulatively, a desiredphenotype. Such methods often are timeand prone to error. In addition,it is often very difficult or impossible to identify the genes that havebeen mutated using such methods.

[0010] Further, methods for making homozygous mutations in culturedcells, where the mutated genes are not known in advance of mutation, arenot known to currently exist. Still further, without a way to identify ahomozygous mutation, the artisan cannot associate the phenotype with agiven mutation. Currently, to associate a desired phenotype with ahomozygous mutation in a cultured cell, the location, structure and/orfunction of the gene must be known to the artisan in advance. Hence, themethods of mutation known in the art are not suitable for homozygouslymutating a cell to achieve a desired phenotype and identifying thegene(s) responsible for the phenotype. Nor are there methods suitablefor making cells with multiple mutations that cumulatively produce adesired phenotype and identifying the genes responsible for thephenotype.

[0011] Several approaches for introducing mutations into eukaryoticgenes are currently in use. Each has significant limitations.

[0012] One approach is homologous recombination to mutate the level ofgene expression or activity of a gene product in a cell. 1: Montgomeryet al., Cell. 1991 Feb 22;64(4):693-702; 2: Riele et al. Nature. 1990Dec 13;348(6302):649-51; 3: Mansour et. al., Proc Natl Acad Sci U S A.1990 Oct;87(19):7688-92; 4: Koller et al., Proc Natl Acad Sci U S A.1989 Nov;86(22):8927-31;5: Capecchi MR. Science. 1989 Jun16;244(4910):1288-92; 6: Zimmer A, Gruss P. Nature. 1989 Mar9;338(6211):150-3; 7: Joyner AL, Skarnes WC, Rossant J. Nature. 1989 Mar9;338(6211):153-6; 8: Thompson S, Clarke AR, Pow AM, Hooper ML, MeltonDW. Cell. 1989 Jan 27;56(2):313-21; 9: Doetschman T, Maeda N, SmithiesO.Proc Natl Acad Sci U S A. 1988 Nov;85(22):8583-7; 10: Doetschman T,Gregg RG, Maeda N, Hooper ML, Melton DW, Thompson S, Smithies O. Nature.1987 Dec 10-16;330(6148):576-8;11: Thomas KR, Capecchi MR. Cell. 1987Nov 6;51(3):503-12.

[0013] Typically, this approach is taken in embryonic stem cells orembryonic germ cells, which are used to make transgenic animals carryingthe mutation of interest. An important limitation of this approach isthat the gene to be mutated must be known in advance of mutation, clonedand sequenced to ensure that the mutagenic vector used in homologousrecombination contains the appropriate targeting sequences. Furthermore,the process is laborious and results in only one mutant copy of the geneof interest in the cell. Where a phenotype depends on homozygosity forexpression, the heterozygous cell, therefore, cannot be used to screenfor a change in a phenotype of interest unless additional work iscarried out to eliminate the second copy of the gene by homologousrecombination. This additional work is time consuming and expensive, andmore importantly can only be done on genes that are known to the artisanin advance.

[0014] Such mutated heterozygous cells can be used to make transgenicanimals. However, such animals will also be heterozygous and may notexpress a phenotype different from the wildtype or nonmutant animal.Further breeding of the animals to homozygosity is therefore required ifone desires to analyze the phenotypic effect of the mutation. Suchbreeding is time consuming and expensive.

[0015] Another approach involves chemical mutagenesis of cells and/ororganisms (see, e.g., Brown et al., Hum. Mol. Genet. 7:1627-1633 (1998);Chen et al., Nature Gen. 24:314-317 (2000); Munroe et al., Nature Gen.24:318-321 (2000); Nolan et al., Nature Gen. 25:440-443 (2000); thedisclosures of all of which are incorporated herein by reference intheir entireties for teaching the use of ENU to generate mutations thatresult in detectable phenotypes in cells or animals). This approachrelies upon the use of one or more chemical mutagens that are able toproduce one or more mutations in the genome. As is the case for mutationby homologous recombination, however, chemical mutagenesis alsotypically results in mutagenesis of only a single copy of a given gene.Since in cases where homozygous mutation is required to achieve adesired phenotype, both copies of a given gene must be mutated before adesired phenotype can be achieved, cells or organisms that undergo asingle round of chemical mutagenesis typically do not show a desiredchange in phenotype. Hence, these cells or organisms generally are notuseful for achieving for a desired phenotype.

[0016] A further problem is that while chemical mutagenesis results inthe mutation of one or more genes in a cell, there is no straightforwardway to determine the mutated gene(s) responsible for the phenotype. Thisapproach also fails to provide a method for making multiple mutationsthat cumulatively provide a desired phenotype that also permits thegenes responsible for the phenotype to be easily identified.

[0017] As discussed above (for homologous recombination mutagenesis)mutated heterozygous cells prepared by chemical mutagenesis can be usedto create transgenic animals. However, the animals will also beheterozygous and may not, therefore, manifest a change in a desiredphenotype from the wildtype. Time-consuming and costly breeding of theanimals to homozygosity is required. Even if a change in the desiredphenotype is observed in the transgenic animals (even in homozygoustransgenic animals), it is very difficult, if not impossible, toidentify the mutated gene(s) responsible for the phenotype. Therefore, alarge number of breedings must be carried out to clone the mutated geneby standard positional cloning methods. Hence, this process is slow,expensive, difficult to carry out on large numbers of mutant animals,and has a high failure rate. Thus, chemical mutagenesis fails to providehomozygous mutations in cultured cells (and hence, in transgenic animalsproduced from such cells) and fails to provide a simple way to identifythe mutated gene(s) responsible for a phenotype in cultured cells or inmulti-cellular organisms.

[0018] Another approach that has been used to mutate genes involves theuse of insertional mutagens, such as gene trap vectors, to mutate genes(e.g., Amsterdam et al., Genes Dev. 13:2713-2724 (1999); von Melcher etal., Genes Dev. 6:919-927 (1992); Gogos et al., J. Virol. 71:1644-1650(1997); Voss et al., Dev. Dyn. 212:171-180 (1998); Zambrowicz et al.,Proc. Natl. Acad. Sci. USA 94:3789-3794 (1997); Friedrich et al., GenesDev. 5:1513-1523 (1991); the disclosures of all of which areincorporated herein by reference in their entireties for teaching theuse of gene traps as a mutagenesis technique). These vectors aretypically inserted into the genome of a cell by non-homologousrecombination. Upon insertion, these vectors are designed to disrupttranscription and/or translation of a gene. Unfortunately, gene trapvectors are inefficient mutagens and mutate only one copy of a givengene. As a result, homozygous mutations typically cannot be created incell culture with such mutagens. In animals, the mutant animal must bebred to homozygosity of the mutant gene prior to phenotypic analysis.Since it is difficult and expensive to breed large numbers of animals tohomozygosity, this approach has only been used on a relatively smallnumber of genes to date.

[0019] This approach also fails to provide a method for making multiplemutations that cumulatively provide a desired phenotype and where thegenes responsible for the phenotype can be identified. The probabilitiesof achieving, in a single cell, insertions in each of the genesrequired, is low and decreases with the number of genes required to bemutated in order to achieve the desired phenotype. Thus, gene traps failto mutate multiple genes and fail to create homozygous mutations inculturedStark et al. (Human Molecular Genetics 8:1925-1938 (1999)), in areview article on forward genetics in mammalian cells with functionalapproaches to gene discovery, suggested a potential alternative tocomplementation of mutants by using expression libraries in order toclone the missing gene. They indicated that the alternative involvesretrovirus-mediated insertional mutagenesis in conjunction with chemicalmutagenesis. They indicated that it was impractical to use insertionalmutagenesis de novo to inactivate two alleles of a target gene. However,there was no indication of how this might be achieved and the referencefailed to disclose a description of the method having been carried outin practice. It was suggested to obtain a population of heavilymutagenized cells and then insert retroviruses into those cells toinactivate and mark the gene. However, there was no report of thissuggestion having been carried out, no guidance regarding how to performeither of the mutagenesis steps to achieve a successful homozygousmutation.

[0020] Accordingly, there exists a need in the art to create homozygousgene mutations on a genome-wide basis in cell culture and inmulticellular organisms without knowledge of the gene in advance. Thereis also a need to provide a way to identify the gene. There is also aneed for a method of mutating multiple genes in a cell, requiredcumulatively to achieve a desired phenotype and to identify one or moreof the mutated genes. The ability to mutate multiple genes or to mutateboth copies of the same gene in cultured cells or multiorganisms,coupled with the ability to identify the mutant gene(s) would be ahighly useful approach to identify novel genes, correlate genes withfunctions, and use the mutant genes, their wildtype counterparts, andother variants, for example, in drug screening and development,transgenic animal and plant production and in the production ofdesirable gene products.

Summary of Invention

[0021] The present invention provides, for the first time, a solution tothe needs identified above by providing methods of efficiently mutatingmultiple genes in the same cell and tagging at least one of the mutatedgenes in cells that contain the mutated multiple genes, so that theidentity of one or more of the mutated genes can be achieved. Thepresent invention also provides methods of making homozygous genemutations and tagging the mutated gene.

[0022] In general terms, the present invention is directed to methodsfor creating mutated cells and multicellular organisms using two or moremutagens where at least one of the mutagens is a polynucleotide thatacts as an insertional mutagen. The polynucleotide can be used as a tagto identify the cell containing the mutated gene and/or to identify themutated gene itself. One aspect of the invention is directed to methodsfor mutating multiple genes, that cumulatively produce a desiredphenotype, within the same cell, and tagging at least one of the mutatedgenes. Another aspect of the invention is directed to methods ofcreating one or more homozygous mutations in a cell, that sufficientlyalter the mutated gene function to generate a desired phenotype, andthat tag at least one of the mutated genes.

[0023] One mutational approach used in accordance with the inventionrelates to methods of mutating cells using physicochemical mutagens.These methods have been shown to efficiently produce mutations in cells,but cannot be used to tag the mutated genes. Since the genes cannot betagged identification of chemically mutated genes is difficult orimpossible.

[0024] The other mutational approach used in accordance with theinvention relates to methods of mutating cells using insertionalmutagens. These methods are known to create heterozygous gene mutationsand, in certain cases, to tag the mutated gene. But these methods havenot been shown to produce homozygous gene mutations within individualcells or to efficiently produce mutation of multiple genes in a cellwhich cumulatively produce a desired phenotype. Thus, the presentinvention utilizes physicochemical mutagenesis in conjunction withinsertional mutagenesis to achieve multiple mutations (that cumulativelyproduce a desired phenotype) within a cell, such that at least one ofthe mutated genes in each cell containing the multiple mutations istagged. Thus, one or more of the mutated genes can be identified bydetecting the tag. In the case of homozygous mutations, at least one ofthe mutated copies of the gene is tagged so that the mutation that isresponsible for the desired phenotype can be identified.

[0025] According to the invention, physicochemical mutagens are used tomutate one or more genes in a cell. Mutations include, but are notlimited to, point mutations, insertions, deletions, inversions, basemodifications and translocations. Such mutations result in events suchas splicing defects that alter exon utilization, frame shifts,truncation (5′ and 3′) of transcripts and of proteins (amino terminaland carboxy-terminal) and alteration of the sequence or abundance of theprotein products of mutated genes by changing the identify of individualresidues in the primary protein sequence, by fusing the coding sequenceof more than one gene to create fusion protein and by changing theabundance of otherwise normal proteins through mutagenesis-inducedchanges in the production, stability or translatability of transcriptsfrom mutated genes.

[0026] Since most eukaryotic genomes are at least diploid (e.g., eachgene consists of at least two copies), when a gene is mutated in aeukaryotic cell in this fashion, only one copy is typically mutated sothat the other copy remains unmutated. Therefore, a homozygous mutation,in which gene expression from both copies of a given gene is eliminated,is not typically achieved by physicochemical mutagenesis alone.

[0027] Therefore, in order to circumvent this deficiency, insertionalmutagenesis is also carried out in addition to the physicochemicalmutagenesis. In this procedure, the level of physicochemical mutagenesisis adjusted to be mutagenic but not to generate the phenotype ofinterest in the absence of the insertional mutagenesis. One or moreinsertional mutagens is inserted into the genome of the host cell ororganism in such a manner so as to alter the expression of a functionalgene product (e.g., an RNA or protein) of one or more cellular genes.The one or more inserted mutagens also have the property of tagging theinsertionally mutated gene, thereby allowing it to be identified.

[0028] By carrying out both physicochemical mutagenesis and insertionalmutagenesis on the same cell, a cell is created in which one or moregenes have been mutated by the physicochemical mutagen and one or moregenes have been mutated by the insertional mutagen. In one suchembodiment of the invention, a cell is created in which one copy of agiven gene has been mutated by the physicochemical mutagen and the othercopy of the same gene has been mutated by the insertional mutagen,thereby creating cells that carry homozygous mutations in that gene.Such mutations can produce desired phenotypes. The mutant cell can thusbe screened for the production of desired phenotypes and the tag can beused to identify the gene responsible for the phenotype.

[0029] Using the two mutational approaches, cells are also provided thatcontain mutations in more than one gene which cumulatively act toproduce a desired phenotype. At least one of the mutated genes thatcontribute to causing the phenotype is tagged by an insertional mutagen.Taking a simple case in which two genes are required to be mutated, theinvention includes the following scenarios (1) Gene #1 is mutated byphysicochemical mutagenesis and Gene #2 is mutated by insertionalmutagenesis in one cell. (2) Gene #1 is mutated by insertionalmutagenesis and Gene #2 is mutated by physicochemical in a second cell.Both cells exhibit the desired phenotype, which is caused by themutation of two genes cumulatively. In the first cell, only Gene #2 canbe identified by the tag. In the second cell, only Gene #1 can beidentified by the tag. These two cells, however, provide completeinformation about the identity of the genes that must be mutated toachieve the desired phenotype.

[0030] The two types of mutational events described herein can becarried out in either order, or simultaneously. The mutational eventscan also be repeated, such that a given cell, population of cells ororganism can be subjected to physicochemical mutagenesis and/orinsertional mutagenesis one or more (e.g., two, three, four, five, six,seven, eight, nine, ten, fifteen, twenty, etc.) times, in any order orsimultaneously. At any point(s) in this process the cells or organismcan be screened for a desired phenotype, or for insertion of aninsertional mutagen both having a desired phenotype or containing theinsertion may be isolated and cloned. In a preferred embodiment,physical chemical mutagenisis proceeds insertional mutagenisis, cellsare selected for insertional mutagenisis. The insertional mutagen is a5" gene trap with signals for site specific recombination andtransposition.

[0031] The invention also provides polynucleotides that act asinsertional mutagens for use in the insertional mutagenesis methods ofthe invention. Such polynucleotides can have any nucleotide sequence andany geometry (e.g., linear, circular, coiled, supercoiled, etc.), andcan be double-stranded or single-stranded. Preferred insertionalmutagens are described in the Figures and Examples herein. The onlylimitation is that they be mutagenic and provide the tagging function.

[0032] In a preferred embodiment, the insertional mutagen comprises asplice acceptor sequence that is not operably-linked to a promotersequence. In other preferred embodiments, the insertional mutagencomprises one or more of the following elements: stop codons in allthree frames found 3′ to the splice acceptor, an internal ribosome entrysite, a selectable marker, and a polyA trap. PolyA traps are describedin detail in U.S. Application No. 09/276,820 herein incorporated byreference for the teaching of polyA traps. In the above embodiment, theselectable marker preferably is operably-linked to a polyadenylationsignal. In other preferred embodiments, the insertional mutagen containsretrovirus sequences that allow the retrovirus replication and infectioncycle. In other preferred embodiments, the insertional mutagen containssequences necessary for transposition. In a highly preferred embodiment,the insertional mutagen contains a splice acceptor that is notoperably-linked to a promoter, the splice acceptor having an optimalbranch point, stop codons in all three frames, an internal ribosomeentry site that includes an exonic splicing enhancer, a selectablemarker with a polyadenylation signal, a polyA trap, and wherein theseelements are contained in a retrovirus vector or contain transpositionsignal sequences. In a further preferred embodiment, the insertionalmutagen contains a splice acceptor not operably-linked to a promoter,the splice acceptor containing an optimal branch point, stop codons inall three frames, an internal ribosome entry site, a selectable markerwith a polyadenylation signal operably-linked to it, and wherein theinsertional mutagen is a retrovirus vector or contains transpositionsignals. Furthermore, in any of the embodiments herein and especially inthe preferred embodiments of above, preferred insertional mutagens alsocontain recombination sites for site specific recombination as describedherein.

[0033] The invention also encompasses cells created by the methods ofthe invention, which preferably are eukaryotic cells (e.g., plant cells,fungal cells (including yeast cells), animal cells (including insectcells, avian cells, worm cells, mammalian cells (including human cells,non-human primate or simian cells, rodent (rat, mouse, etc.) cells,rabbit cells, bovine cells, ovine cells, porcine cells, canine cells,feline cells), and the like). These cells can be isolated and cloned,using methods that are well-known in the art to those of ordinary skill.Primary or established cells can be used in the methods of the inventionto prepare the mutated cells.

[0034] In another aspect, the invention also provides libraries of cellscreated by the present invention. A physicochemical/insertional mutationlibrary is created when both mutational processes are carried out onmore than one cell, preferably 10⁶-10⁸ cells (for cells with largegenomes such as mammalian cells) or as needed to ensure mutationalsaturation with the combined mutagenesis. The population of cells canall be the same (as in a cell line), or can comprise differentsubpopulations (as, for example, in cell populations prepared fromtissues). Each clone in such libraries may contain a set of mutatedgenes that is distinct from the set of mutated genes in other cloneswithin the library. Alternatively, the same genes may be mutated indifferent clones but the type of mutation of each gene could bedifferent. For example, all three genes, Genes #1, #2, and #3, could bemutated in three different clones but the type of mutation (insertionalvs physicochemical) could differ. (See schematic) Such libraries ofcells therefore, are useful to rapidly screen for desired phenotypes(e.g., changes from the wildtype or nonmutant phenotype) that resultfrom various single mutations or various combinations of mutations.

[0035] A library can also encompass a population (two or more,preferably 10²-10⁵ of cells that has been subjected to eitherinsertional mutagenesis or physicochemical mutagenesis but not both.These libraries serve as a population of cells that form a substrate forfurther mutation by the second mutagenic process of the invention. Theefficiency of physicochemical mutagenesis permits the creation ofsmaller libraries that contain mutation in all genes than are obtainedusing the less efficient insertional mutagenesis.

[0036] Accordingly, the invention encompasses a method for making alibrary of mutagenized cells by insertionally mutagenizing at least onephysicochemically mutagenized cell from among two or morephysicochemically mutagenized cells. The invention is also directed to amethod for making a library of mutagenized cells by physicochemicallymutagenizing at least one insertionally mutagenized cell from among twoor more insertionally mutagenized cells. The invention also encompassesmethods for making a library of mutagenized cells by simultaneouslymutagenizing two or more cells with one or more physicochemical mutagensand one or more insertional mutagens. The invention also encompassesmethods for making a library of mutagenized cells by subjecting two ormore cells containing a physicochemical mutagen to insertionalmutagenisis. The invention is also directed to a method for making alibrary of mutagenized cells by administering a physicochemical mutagento two or more cells containing an insertional mutagen. One or morecells can be screened for mutation of a specific desired gene or otherdesired phenotype at any stage of the process, such as after anyphysicochemical mutagenesis event, after any insertional mutagenesisevent, or after both or all mutagenesis events. Cells having a desiredphenotype can be isolated and cloned. Cells can also be screened for thepossession of an insertional mutagen at any stage of the process, suchas after any physicochemical mutagenesis event, after any insertionalmutagenesis event, or after both or all mutagenesis events. These cellscan be subjected to further insertional mutagenesis or physicochemicalmutagenesis. These cells can be isolated and/or cloned at any stage ofthe process, such as after selection for possession of an insertionalmutagen. Such clones can accordingly form the substrate for furthermutagenesis events. In one embodiment, physicochemical mutagenesis isperformed on a plurality of cells. The cells are then expanded to formclonal populations and these populations are subjected to insertionalmutagenesis so that a heterogeneous population of insertionallymutagenized cells is formed. These cells may or may not be expanded butcan be screened for mutation of a specific desired gene or for a desiredphenotype. In another exemplary embodiment, a plurality of cells isphysicochemical mutated, the cells expanded, and then insertionallymutagenized. This first insertion may or may not be the insertion thatcauses the phenotype. An insertion that causes the phenotype may beproduced from further insertions of the original insertional mutagen.For example, after the original insertional mutagenesis event, the cellscould be expanded and, after expansion, a component could be introducedinto the cell or induced in the cell that will cause further insertionalmutagenesis, such as a transposase. In a related exemplary embodiment,cells can be both physicochemically mutagenized and insertionallymutagenized (simultaneously) expanded, and a factor to cause insertionalmutagens can be introduced either into the cells or can be induced inthe cells. In a related exemplary embodiment, cells can be insertionallymutagenized with the introduction of a first insertional mutagen,expanded, and then a factor causing further insertion from theendogenous insertional mutagen can be activated in the cells which canthen be physicochemically mutated. As mentioned, cells may or may not beexpanded following each treatment.

[0037] At any stage after a phenotype is produced by mutation, thepresence of site-specific recombination sequences on the insertionalmutagen can be used to ascertain whether the insertion caused thephenotype. If the excision of the insertional mutagen reverts thephenotype, this indicates that mutation causing the phenotype was causedby an insertion.

[0038] The invention also provides methods of using the cells andlibraries to screen for phenotypes that are created by the mutagenesismethods of the invention and to identify one or more mutationsresponsible for the phenotype.

[0039] The methods of the invention provide a way to establish thefunction of a gene. With the methods it is possible to determine thefunction of any specific desired gene. Cells can be mutated and screenedfor a mutation in a specific desired gene with any assay that can beused to specifically detect a mutation in that gene. The effect of themutation on the cell or animal can then be ascertained. In this case,the tagged insertional mutagen is useful for identification of cellscontaining mutation of the gene of interest.

[0040] Alternatively, mutated cells can be screened at random for adesired phenotype, or for production of a desired phenotype in amulti-cellular organism made from the cell, and the phenotype can bethen correlated with mutation in one or more genes by means of the tag.Alternatively, a cell that is mutated can be selected on the basis ofthe phenotype it has or confers on an organism made from the cell, andthat phenotype can then be correlated with mutation in one or more genesby means of the tag. Thus, any change in phenotype of the cell (or ofmulticellular organism derived from the cell) from that of thenon-mutated cell (or cellular organism) can be ascribed to the mutatedgene. Mutated genes that give rise to desired phenotypes can beselected, identified, and characterized e.g., cloned, sequenced, mapped,etc. According to this aspect of the invention, the function of any genecan be identified and assessed. Thus, a phenotype can be correlated witha gene that is known in the art (previously identified, e.g., mapped,cloned, sequenced, or otherwise characterized) or with a gene that isnot known in the art.

[0041] Although the methods of the invention identify a mutated gene,the invention provides a way to correlate the gene with a function andthus provides a way to ascribe a function to the wildtype gene and touse that wildtype gene and gene product. The invention, therefore,provides for use of the wildtype gene or other natural variant of thegene that is identified as described above. This includes, but is notlimited to, allelic variants, homologs, orthologs, pseudogenes, and thelike. The wildtype gene, that has been identified by means of themutated version, as well as other variants, can be isolated, forexample, from non-mutated cells, using standard recombinant DNA ormolecular biological techniques, such as cDNA library screening or PCR.The wildtype gene/protein or other variant can be used, for example, asa therapeutic protein, antibody target. Naturallymutants are also usefulas therapeutic or diagnostic targets, for example, with antibodies orother detectable and/or inhibiting binding reagent.

[0042] In a diseased tissue or cell, a naturally mutant gene gives riseto the disease. Further mutation by the present method can revert thecell or organism to a normal phenotype allowing identification of themutated disease gene.

[0043] The invention also encompasses use of the mutated cells toproduce transgenic animals. Transgenic animals can be created frommutant somatic or germ cells, or from mutant stem cells (e.g., embryonicor adult stem cells), that have been produced by methods of theinvention. Donor cells (which may be a somatic cell, an adult stem cell,a germ cell or an embryonic stem cell from a donor animal) are subjectedto insertional mutagenesis and physicochemical mutagenesis in vitro soas to produce a mutated donor cell with a single homozygous mutationthat produces a desired phenotype in the cell or organism or a mutateddonor cell with mutations in multiple genes (that cumulatively willachieve a desired phenotype in the cell or organism). The animal can bemade by transferring the nucleus from the donor cell to a recipient cell(which may be, for example, a fertilized oocyte that has beenenucleated), and producing a transgenic organism from the recipientcell. Alternatively, the mutant stem cell could be implanted into ablastocyst or the mutant germ cell used to create a mutant zygotethrough in vitro fertilization or artificial insemination and theresulting mutant zygotes put into a pseudo-pregnant female to producethe transgenic organism.

[0044] Genetically modified animals can be created by transplantation ofnuclei from cells that have been mutagenized by the techniques of thepresent invention. Nuclei extracted from mutant cells are then implantedinto enucleated fertilized eggs, and the resultant zygote is implantedinto a pseudopregnant female to develop into an animal carrying themutations that were generated in the original mutagenized cell.

[0045] Zygotes can also be formed from mutant embryonic or otherpluripotent stem cell following the blastocyst fusion protocols thathave been developed for the creation of genetically modified mice.

[0046] Briefly, the modified stem cells are combined with cells of adiploid or tetraploid morula or the modified cells are injected directlyinto the blastocoel of a developing blastocyst. The chimeric zygote thatresults is implanted into a pseudopregnant female to develop into ananimal carrying the mutations generated in the stem cell. Geneticallymodified germ cells can be created by in vitro retroviral-mediated orother gene delivery into spermatogonial stem cells of both adult andimmature animals and can result in stable integration of the insertionalmutagen in 2-20% of stem cells. After transplantation of the transducedstem cells into the testes of infertile recipient animals, approximately4.5% of progeny from these males contain the insertional mutagen, andthis mutagenic vector is transmitted to and functions in subsequentgenerations. 1: Chesne P, Adenot PG, Viglietta C, Baratte M, BoulangerL, Renard JP.Nat Biotechnol. 2002 Apr;20(4):366-9; 2: Hosaka K, Ohi S,Ando A, Kobayashi M, Sato K. Hum Cell. 2000 Dec;13(4):237-42; 3: Wolf E,Zakhartchenko V, Brem G. Biotechnol. 1998 Oct 27;65(2-3):99-110.; InHogan B, Beddington R, Costantini F, Lacy E. Manipulating the MouseEmbryo; a Laboratory Manual Cold Spring Harbor Laboratory Press. 1994;1: Cecconi F, Gruss P.Methods Mol Biol. 2002;185:335-46. Review; 1:Brinster RL. Science. 2002 Jun 21;296(5576):2174-6; 2: Nagano M,Brinster CJ, Orwig KE, Ryu BY, Avarbock MR, Brinster RL. Proc Natl AcadSci U S A. 2001 Nov 6;98(23):13090-5; 1: Cecconi F, Gruss P. Methods MolBiol. 2002;185:335-46.

[0047] In another embodiment, transgenic organisms can be created fromcells chosen because they display a desired phenotype in vitro afterbeing subjected to insertional and physicochemical mutagenesis accordingto the methods of the invention. The identity of the gene or genesresponsible for the phenotype may or may not be known at this stage forthe preparation of transgenic animals. In another embodiment transgenicorganisms can be created from a cell with a mutation in a specificdesired gene. In another embodiment, mutated cells can be selected atrandom, used to make the transgenic organism, and the transgenicorganism can be screened for a desired phenotype.

[0048] The invention also encompasses methods for making transgenicanimals and transgenic animals produced by the present methods.Transgenic animals that can be produced by the methods of the inventioninclude, for example, insects (including Drosophila, Spodoptera andTrichoplusa species), birds, worms (including C. elegans), fish(including zebrafish), mammals (including humans, and non-human mammalssuch as simians and other non-human primates, mice, rats, pigs, cows,sheep, dogs, cats, and the like). The transgenic animals can be used,for example, as models for human disease, to study gene function, toscreen for phenotypes of interest, for agricultural applications, or fordrug testing.

[0049] In one aspect of the invention, transgenic animals are producedfrom cells that have been physicochemically mutated in vitro. The cellsof the recipient animal may then be mutated by insertional mutagenesis.The insertional mutagen can be introduced exogenously into the animal orcan be induced or activated from an endogenous incorporated insertionalmutagen. In one embodiment, the insertional mutagenesis is achieved bycrossing the transgenic animal produced from a physicochemically mutatedcell with another animal containing an insertional mutagen in its germline, such as a transposon, such as those described herein. The animalcan be screened for mutation of a specific desired gene or forexpression of another desired phenotype after the physicochemicallymutated cell has been used to form the animal and/or after theinsertional mutagenesis.

[0050] The invention also provides methods of producing transgenicplants, and transgenic plants produced by such methods. Transgenicplants that can advantageously be produced according to such methodsinclude dicotyledenous and monocotyledenous plants.

[0051] The invention also encompasses the use of the mutant cells fordrug screening. In this embodiment, mutant cells are exposed to testcompounds or compositions which may have therapeutic potential, todetermine the effect of the compound or composition on a desiredphenotype induced by one or more mutations in the mutant cells,including the level of expression or activity of the mutated gene orprotein. Furthermore, the wildtype genes or other variants thatcorrespond to the mutated gene or genes, also can be used to identifydrugs that affect a phenotype caused by the gene or genes, including thelevel of expression or activity of the wildtype or variant gene orprotein of interest.

[0052] Other preferred embodiments of the present invention will beapparent to one of ordinary skill in light of what is known in the art,in light of the following drawings and description of the invention, andin light of the claims.

Brief Description of Drawings

[0053]Figures 1A-1J:Non-limiting examples of 5' gene trap insertionalmutagens vectors useful in the present invention. Each insertionalmutagen is illustrated schematically in its linear form; however,insertional mutagens of the invention can have any geometry (linear,circular, coiled, supercoiled, etc.). Horizontal lines and boxesindicate polynucleotides, such as DNA or RNA. Stop codons can be presentin any reading frame, or nested such that they are present in allreading frames. S/A represents a splice acceptor site. S/D represents asplice donor site. pA represents a polyadenylation signal. ET representsan epitope tag.

[0054]Figures 2A-2H:Non-limiting examples of 5' gene trap insertionalmutagens useful in the present invention. Each insertional mutagen isillustrated schematically in its linear form. Horizontal lines and boxesindicate polynucleotides such as DNA or RNA. SM and RG representselectable marker and reporter gene, respectively. S/A represents asplice acceptor site. S/D represents a splice donor site. pA representsa polyadenylation signal. IRES represents internal ribosomal entry site.

[0055]Figures 3A-3B:Non-limiting examples of 5' gene trap insertionalmutagens useful in the present invention. Each insertional mutagen isillustrated schematically in its linear form. Horizontal lines and boxesindicate polynucleotides such as DNA or RNA. SM and RG representselectable marker and reporter gene, respectively. S/A represents asplice acceptor site. pA represents a polyadenylation signal. IRESrepresents internal ribosomal entry site.

[0056]Figures 4A-4C:Non-limiting examples of 5' gene trap insertionalmutagens useful in the present invention. Each insertional mutagen isillustrated schematically in its linear form. Horizontal lines and boxesindicate polynucleotides such as DNA or RNA. β-geo is a fusion of theneomycin resistance gene and β-galactosidase gene. S/A represents asplice acceptor site. pA represents a polyadenylation signal. IRESrepresents internal ribosomal entry site. 5' LTR and 3' LTR representretroviral long terminal repeats. ψ represents retroviral packagingsignal. In Figure 4B, the 5' LTR, 3' LTR, and ψare shown upside down toindicate that the retroviral sequence is in reverse orientation relativeto the splice acceptor site and β-geo gene. In Figure 4C, the solidboxes represent transposon signals.

[0057]Figures 5A-5E:Non-limiting examples of 5' gene trap insertionalmutagens containing a 3' gene trap component. Each insertional mutagenis illustrated schematically in its linear form. Horizontal lines andboxes indicate polynucleotides such as DNA or RNA. Arrows representpromoters. SM and RG represent selectable marker and reporter gene,respectively. β-geo is a fusion of the neomycin resistance gene andβ-galactosidase gene. S/A represents a splice acceptor site. S/Drepresents a splice donor site. pA represents a polyadenylation signal.IRES represents internal ribosomal entry site. 5' LTR and 3' LTRrepresent retroviral long terminal repeats. ψrepresents retroviralpackaging signal. In Figure 5D, the 5' LTR, 3' LTR, and ψare shownupside down to indicate that the retroviral sequence is in reverseorientation relative to the splice acceptor site and β-geo gene. InFigure 5E, the solid boxes represent transposon signals.

[0058]Figures 6A-6C:Proposed mechanism of gene mutation using a 5' genetrap. Vector is illustrated schematically in its linear form. Horizontallines and boxes indicate polynucleotides such as DNA or RNA. Arrowsrepresent promoters. β-geo is a fusion of the neomycin resistance geneand β-galactosidase gene. S/A represents a splice acceptor site. pArepresents a polyadenylation signal. IRES represents internal ribosomalentry site. Figure 6A shows the insertional mutagen and the endogenousgene prior to vector insertion. Figure 6B shows the endogenous genefollowing insertion of the insertional mutagen. Figure 6C shows thefusion mRNA produced from the endogenous gene.

[0059]Figures 7A-7B:Non-limiting examples of 5' gene trap insertionalmutagens containing a promoter linked to a selectable marker or reportergene followed by a polyadenylation signal. Each insertional mutagen isillustrated schematically in its linear form. Horizontal lines and boxesindicate polynucleotides, such as DNA or RNA. Arrows representpromoters. SM and RG represent selectable marker and reporter gene,respectively. β-geo is a fusion of the neomycin resistance gene andβ-galactosidase gene. S/A represents a splice acceptor site. pArepresents a polyadenylation signal. IRES represents internal ribosomalentry site. 5' LTR and 3' LTR represent retroviral long terminalrepeats. ψrepresents retroviral packaging signal. In Figure 7B, the 5'LTR, 3' LTR, and ψ are shown upside down to indicate that the retroviralsequence is in reverse orientation relative to the splice acceptor siteand β-geo gene. The presence of the promoter operably linked to aselectable marker/reporter gene and polyadenylation signal allowsselection of integrated insertional mutagens independent of whether ornot integration has occurred in a transcriptionally active region of thegenome. The figure is illustrative of several insertional mutagen typescontaining selectable markers; however, the promoter/selectablemarker/polyadenylation signal unit can be used on any of the insertionalmutagens described herein.

[0060]Figures 8A-8C:Non-limiting examples of 5' gene trap insertioninsertional mutagens containing site-specific recombination signals.Each insertional mutagen is illustrated schematically in its linearform. Horizontal lines and boxes indicate polynucleotides such as DNA orRNA. Arrows represent promoters. Filled triangles representsite-specific recombination signals. The site-specific recombinationsignals can be in any orientation relative to one another. The figuredepicts an orientation that promotes excision of the insertional mutagenfrom the genome. If the signals are placed on the insertional mutagen inthe opposite direction relative to each other, the insertional mutagenwould be inverted in the genome following site specific recombination.N-SM represents a negative selectable marker gene. β-geo is a fusion ofthe neomycin resistance gene and β-galactosidase gene. S/A represents asplice acceptor site. pA represents a polyadenylation signal. IRESrepresents internal ribosomal entry site. 5' LTR and 3' LTR representretroviral long terminal repeats. ψrepresents retroviral packagingsignal. In each example, the 5' LTR, 3' LTR, and ψ are shown upside downto indicate that the retroviral sequence is a reverse orientationrelative to the splice acceptor site and β-geo gene. The position of thesite-specific recombination signals in figures 8A and 8C is shown in theviral LTRs such that most of the viral insertional mutagen can beexcised (see, e.g., Ishida, Nucl. Acids Res. 27: e35 (1999)). Thepresence of the promoter operably linked to a negative selectable markerand polyadenylation signal allows selection for cells in which theintegrated insertional mutagen has been excised. The present figure isillustrative of several insertional mutagen types containingsite-specific recombination signals; however, the site-specificrecombination signals can be used on any of the insertional mutagensdescribed herein, including the non-viral insertional mutagens.

[0061]Figures 9A-9H:Non-limiting examples of 5' gene trap insertionalmutagens containing site-specific recombination signals (alsoequivalently referred to herein as recombination sites). Eachinsertional mutagen is illustrated schematically in its linear form(although insertional mutagens can exist in any conformation, includinglinear, circular, coiled, supercoiled, branched, etc.). Horizontal linesand boxes indicate polynucleotides such as DNA or RNA. Arrows representpromoters. Filled triangles represent site-specific recombinationsignals. The site-specific recombination signals can be in anyorientation relative to one another. Recombination sites shown inopposite orientation relative to one another (e.g., Figs. 9D-9F and 9H)produce an inversion following recombination, whereas recombinationsites shown in the same orientation relative to one other (e.g., Figs.9A-9C and 9G) produce a deletion upon recombination. N-SM represents anegative selectable marker gene, while P-SM represents a positiveselectable marker gene. Neo represents a neomycin resistance gene. TKrepresents a herpesvirus thymidine kinase (HSV-TK) gene. S/A representsa splice acceptor site. pA represents a polyadenylation signal. IRESrepresents an internal ribosome entry site. The presence of the promoteroperably linked to a negative selectable marker and polyadenylationsignal allows selection for cells in which the integrated insertionalmutagen has been excised. Where the positive selectable marker and/orthe negative selectable marker lack a promoter on the insertionalmutagen, the marker can be expressed from an endogenous promoter uponintegration of the insertional mutagen into the genome of the host cell.The present figure is illustrative of several insertional mutagen typescontaining site-specific recombination signals; however, therecombination sites can be used on any of the insertional mutagensdescribed herein, including the non-viral insertional mutagens. Inaddition, any of the insertional mutagens shown in this figure canoptionally contain no (or only one) site-specific recombinationsignal(s). The insertional mutagens depicted in Figures 9A-9F canoptionally lack the S/A, IRES and/or pA signal. Each of the insertionalmutagens shown optionally can be configured as a viral insertionalmutagen and therefore can contain 5' and 3' LTRs and packaging signals.As one of ordinary skill will readily appreciate, other insertionalmutagen elements described herein and/or recognized in the art can beincluded in the insertional mutagens in addition to the elementsillustrated in the figures.

[0062]Figures 10A-10G:Non-limiting examples of 5' gene trap insertionalmutagens containing multiple exons. Each insertional mutagen isillustrated schematically in its linear form (although vectors can existin any conformation, including linear, circular, coiled, supercoiled,branched, etc.). Horizontal lines and boxes indicate polynucleotidessuch as DNA or RNA. Arrows represent promoters. S/A represents a spliceacceptor site, and S/D represents a splice donor site. pA represents apolyadenylation signal. IRES represents an internal ribosome entry site.SM indicates a positive or negative selectable marker. A reporter genecan be substituted for the SM on any of the insertional mutagens shownin this figure. In Fig. 10F and 10G, the selectable marker open readingframe has been separated onto different exons. Upon transcription froman endogenous gene, followed by splicing, the open reading frame will bereconstituted to produce a functional SM. It will be recognized by theordinarily skilled artisan that each of the insertional mutagensdepicted in this figure can optionally contain one or more site-specificrecombination signals (see Figure 9). Optionally, the insertionalmutagens depicted in this figure can lack the S/A, IRES, S/D and/or pAsignal. Each of the insertional mutagens shown optionally can beconfigured as a viral insertional mutagen and therefore can contain 5'and 3' LTRs and packaging signals. As one of ordinary skill will readilyappreciate, other elements described herein and/or recognized in the artcan be included in the insertional mutagens in addition to the elementsillustrated in the figures.

[0063]Figure 11:Method for detecting gene trap insertions that occur indevelopmentally regulated genes. In this example, cells are identifiedin which a transcriptionally active gene became down regulated orsilenced in response to specific treatments or environmental stimuli tothe cells. DNA is illustrated schematically in its linear form.Horizontal lines and boxes indicate polynucleotides such as DNA or RNA.Arrows represent promoters. Filled triangles represent site-specificrecombination signals. The site-specific recombination signals aredepicted in an orientation that promotes excision of the positiveselectable marker from the genome. P-SM and N-SM represent positiveselectable marker and negative selectable marker, respectively. S/Arepresents a splice acceptor site. S/D represents a splice donor site.pA represents a polyadenylation signal. IRES represents internalribosomal entry site.

[0064]Figure 12:Method for detecting gene trap insertions that occur indevelopmentally regulated genes. In this example, cells are identifiedin which a transcriptionally silent gene (or minimally expressed gene)became turned on or enhanced in response to specific treatments orenvironmental stimuli to the cells. DNA is illustrated schematically inits linear form. Horizontal lines and boxes indicate polynucleotidessuch as DNA or RNA. Arrows represent promoters. Filled trianglesrepresent site-specific recombination signals. The site-specificrecombination signals are depicted in an orientation that promotesexcision of the negative selectable marker from the genome. P-SM andN-SM represent positive selectable marker and negative selectablemarker, respectively. S/A represents a splice acceptor site. S/Drepresents a splice donor site. pA represents a polyadenylation signal.IRES represents internal ribosomal entry site.

[0065]Figure 13:Method for detecting gene trap insertions that occur indevelopmentally regulated genes. In this example, cells are identifiedin which a transcriptionally silent gene (or minimally expressed gene)became turned on or enhanced in response to specific treatments orenvironmental stimuli to the cells. In other examples, it is possible toidentify cells in which a transcriptionally active gene became downregulated or silenced in response to specific treatments orenvironmental stimuli to the cells. This is accomplished using thevector shown in this figure in combination with the selection schemeshown in figure 11 (i.e. selection for the positive selectable marker,then treatment of cells with an agent capable of altering its expressionpattern, and selecting against cells expressing the negative selectablemarker). DNA is illustrated schematically in its linear form. Horizontallines and boxes indicate polynucleotides such as DNA or RNA. Arrowsrepresent promoters. Filled triangles represent site-specificrecombination signals. The site-specific recombination signals aredepicted in an orientation that promotes inversion of the positive andnegative selectable markers within the genome. P-SM and N-SM representpositive selectable marker and negative selectable marker, respectively.S/A represents a splice acceptor site. S/D represents a splice donorsite. pA represents a polyadenylation signal. IRES represents internalribosomal entry site.

[0066]Figure 14:The Figure schematically shows non-limiting examples ofcells that result from mutagenesis according to the present inventionand examples of how genes could be tagged for detection. X denotes aphysicochemical event. T denotes a tag introduced by insertionalmutagenesis event.

[0067] (A) Phenotype results from homozygous mutation of single gene.Both copies of gene A contain a mutation. The gene can be identified bythe tag on one copy. Only one cell is required to identify the generesponsible for the phenotype (I). Cell II can be discarded.

[0068] (B) Phenotype results from heterozygous mutation of two differentgenes. Cell I allows identification, in the same cell, of the two genesresponsible for the phenotype. Cells II and III are used in combinationto identify both genes responsible for the phenotype or separately toidentify one of the genes responsible for phenotype. Cell IV can bediscarded.

[0069] (C) Phenotype results from heterozygous mutation of Gene A andhomozygous mutation of Gene B. Cell I allows identification, in the samecell, of the two genes responsible for the phenotype. Cells II and IIIare used in combination to identify both genes responsible for thephenotype or separately to identify one of the genes responsible forphenotype. Cell IV can be discarded.

[0070] (D) Phenotype results from homozygous mutation of two differentgenes. Cell I allows identification, in the same cell, of the two genesresponsible for the phenotype. Cells II and III are used in combinationto identify both genes responsible for the phenotype or separately toidentify one of the genes responsible for phenotype. Cell IV can bediscarded.

[0071]

[0072]

[0073]Figure 17: A schematic representation of a combined mutagenesisscreen to identify genes needed for FasL induced apoptosis.

[0074]Figure 18: A schematic representation of the Fas induced apoptosispathway. To identify gene traps that affect sensitivity to FasL, thedoubly mutagenized combined mutagenesis library was selected for clonesthat exhibited resistance to Fas-induced apoptosis. Under the FasLselection conditions, there is a low spontaneous backgroundFigure 19:Cre mediated excision of combined mutagenesis gene trap reversion ofFas-resistant phenotype. Clones showing FasL resistance caused by thepDKO2 gene trap were analyzed using RT-PCR, 5"-RACE and inverse PCR toidentify the biologically active trapped genes.

Detailed Description

[0075]DefinitionsIn the description that follows, a number of terms usedin recombinant DNA technology are utilized extensively. In order toprovide a clear and consistent understanding of the specification andclaims, including the scope to be given such terms, the followingdefinitions are provided.

[0076] As used herein, the term physicochemical mutagenesis means anymethod of mutating genes that is not insertional mutagenesis (i.e., byinsertional mutagen as defined below), such as ionizing radiation and/orchemical approaches to induce one or more mutations in a cell ororganism. Physicochemical mutagenesis, therefore, encompasses use ofchemical mutagens, radiation (e.g., UV, αradiation, βradiation, γradiation, x-rays), error prone replication proteins (for example,without limitation, mutant DNA polymerases, such as those that lack aproofreading function), restriction enzymes (used to create DNA breaksand deletions upon introduction into a host cell), and DNA repairmutants and inhibitors (used to enhance mutation from spontaneous andinduced mutation). Examples include, but are not limited to, mutant celllines defective for the genetic complementation groups of xerodermapigmentosum (XP) known as XP-G and XP-A, the DNA repair protein Ku, andthe DNA-dependent protein kinase DNA-PK. An example of a DNA repairinhibitor is O⁶-BG, an inhibitor of O⁶-alkylguanine-alkyltransferase.Any physicochemical mutagen can be used alone or in combination with oneor more other physicochemical mutagens.

[0077] Insertional mutagenesis, as it relates to the invention, means aprocess in which a polynucleotide is inserted into the genome of a cellin such a way so as to mutate an endogenous gene. As used herein theterms incorporation or integration or insertion into an endogenous geneare used synonymously.

[0078] Insertional mutagenesis can occur when an insertional mutagen isintroduced into a cell exogenously and as a result of the exogenousintroduction becomes incorporated into the genome so as to mutate one ormore endogenous genes. The invention, however, is also directed tomutagenesis events that occur when an endogenous insertional mutagen iscaused to insert into locations that are different from the originallocation. Such is the case when an endogenous transposable element whichis induced to further transposition by the action of a transposase.Accordingly, in one embodiment of the invention, insertional mutation ofan allele or a gene results from transposition of an endogenousinsertional mutagen. This endogenous insertional mutagen may benaturally-occurring in the cell or may have been introduced into thecellular genome or the genome of a precursor cell such as a precursorcell in vitro or precursor cell in vivo.

[0079] In one aspect of simultaneous introduction of different mutagensto a cell, one or more of the mutagens is produced endogenously. One ormore mutagens is present in the genome of the cell and can provide forfurther insertion into the genome at one or more new locations. Thus,simultaneous mutagenesis can occur by causing the new insertions of oneor more different mutagens from within the cell and can also occur whenthis endogenous introduction is concurrent in time with the introductionof an exogenous mutagen.

[0080] The term gene disruption as used herein refers to a geneknock-out or knock-down in which an insertional mutagen is integratedinto an endogenous gene thereby resulting expression of a fusiontranscript between endogenous exons and sequences in the insertionalmutagen.

[0081] The mutation can result in a change in the expression level of agene or level of activity of a gene product. Activity encompasses allfunctions of a gene product, e.g. structural, enzymatic, catalytic,allosteric, and signaling. In one embodiment, mutation results in adecrease or elimination of gene expression levels (RNA and/or protein)or a decrease or elimination of gene product activity (RNA and/orprotein). Most mutations will decrease the activity of mutated genes.However, both the insertional and physicochemical mutagens can also actto increase or to qualitatively change (e.g. altered substrate onbinding specificity, or regulation of protein activity) the activity ofthe product of the mutated gene. Although mutations will often generatephenotypes that maybe difficult to detect, most phenotypicallydetectable mutations change the level or activity of mutated genes inways that are deleterious to the cell or organism.

[0082] The insertional mutagens can also be used to tag the mutated geneat least at the DNA level, and in one embodiment, at the RNA or proteinlevel, depending on the insertional mutagen. The mutagenic sequence mayby itself be detectable so as to tag the insertionally mutated gene orproduct of the gene.

[0083] Thus, as used herein, the term tag refers to a structural orfunctional feature (typically, a nucleotide sequence) contained on aninsertional mutagen of the invention, which permits the location of theinsertional mutagen to be determined once it has been inserted into atarget nucleic acid molecule via recombination (e.g., into the genome ofa target cell). The tag, accordingly, not only enables location of theinsertional mutagen. It enables the locus into which the vector hasinserted to be identified. Examples of tags include, but are not limitedto, nucleotide sequences encoding a reporter gene (e.g., β-lactamase,β-galactosidase, luciferase, chloramphenicol acetyl transferase, greenfluorescent protein and its derivatives, yellow fluorescent protein andits derivatives, blue fluorescent protein and its derivatives, cyanfluorescent protein and its derivatives, red fluorescent protein and itsderivatives, and the like), and nucleotide sequences encoding aselectable marker (which may be a positive selectable marker or anegative selectable marker). It should be recognized however that a tag,for purposes of the invention need not encode a protein. It simplyprovides a sequence that allows detection of either the tag itself or ofa nucleotide sequence adjacent to or otherwise linked to the taggedsequence.

[0084] The tag can also provide the property that the insertionalmutagen can be detected such that a cell containing the insertionalmutagen can be detected (and isolated, if desired, or otherwisespecifically manipulated).

[0085] 5′ gene traps are insertional mutagens that are designed toprevent or reduce functional expression of a given endogenous gene uponinsertion of the mutagen into the gene. 5" gene traps alter geneexpression by interrupting the normal splicing or exon structure of theprimary transcripts of mutated genes. Splicing interruption isaccomplished by splicing of upstream exons of the mutated gene ontosplice acceptor sequences within the insertional mutagen. This change insplicing often disrupts the protein coding of transcripts of the mutatedgene. Alternatively, the insertional mutagen can insert directly intoand disrupt the coding potential of exons of the mutated gene.

[0086] 3′ gene traps are insertional mutagens that are designed toactivate or otherwise enhance transcription of, and optionallytranslation of, endogenous exons of the gene in which the mutagenicvector is inserted. 3" gene traps function by initiating transcriptionof mutated genes at promoter sequences located within the insertionalmutagen. Transcripts initiated in the mutagenic vector continue 3" ofthe insertion site to include downstream exons of the mutated gene inchimeric primary transcripts. Exons containing vector sequence cansplice onto exons of the mutated gene to generate chimeric transcriptsthat encode proteins including all or C-terminal fragments of the normalprotein product of the mutated gene.

[0087] As used herein, decrease means that a given gene has been mutatedsuch that the level of gene expression or level of activity of a geneproduct in a cell or organism is reduced from that observed in thewildtype or non-mutated cell or organism. This is often accomplished byreducing the amount of mRNA produced from transcription of a gene, or bymutating the mRNA or protein produced from the gene such that theexpression product is less abundant or less active.

[0088] As used herein, and unless otherwise indicated, a means one ormore.

[0089] As used herein, the term mutated clone refers to one or moreprogeny cells arising from a mutated parent cell created with one orboth mutagenesis methods of the invention.

[0090] As used herein, homozygous mutant refers to a cell in which allcopies (typically two in most eukaryotic cells, although certainfilamentous yeasts, and some higher eukaryotic cell lines, may have morethan two copies) of a given gene are mutated.

[0091] As used herein, copies of genes are also known in the art asalleles. This latter term signifies the naturallycopy of a given gene ina cell. Usually there are two copies of a gene in a diploid cell. Insome situations (e.g., trisomy 21) there are three copies, but four ormore copies of one or more entire chromosomes, or extra copies of genesor chromosomal fragments are also encountered naturally. Cells may alsobe experimentally altered to change the number or expression of specificgenes.

[0092] The methods and the compositions of the invention may involveinsertional mutagens that contain target nucleotide sequences forhomologous recombination. As used herein a target sequence allowshomologous recombination of an insertional mutagenic nucleotide withcellular DNA at a predetermined site on the cellular DNA, the sitehaving homology for sequences in the insertional mutagen, the homologousrecombination at the predetermined site resulting in the introduction ofthe insertional mutagen into the genome and subsequent mutation. Atarget sequence may have homology where the sequence or sequences withinthe gene to be mutated or upstream or downstream of the gene to bemutated. The use of targeting sequences has been disclosed in many U.S.patent applications, including U.S. 5,641,670, 6,270,989, and 5,733,761,all incorporated by reference for teaching a target sequence.

[0093] As used herein, non-homologous recombination (which may also bereferred to equivalently as illegitimate recombination) means thejoining (exchange or redistribution) of genetic material through amechanism that does not involve homologous recombination (e.g.,recombination directed by sequence homology) and that does not involvesite-specific recombination (e.g., recombination directed bysite-specific recombination signals and a corresponding site-specificrecombinase). Examples of non-homologous recombination includeintegration of exogenous DNA into chromosomes at non-homologous sites,chromosomal translocations and deletions, DNA end joining, double strandbreak repair, bridge-break-fusion, concatemerization of transfectedpolynucleotides, retroviral insertion, and transposition. In most cases,nonrecombination is thought to occur through the joining of free DNAends. Free ends are DNA molecules that contain an end capable of beingjoined to a second DNA end either directly, or following repair orprocessing. The DNA end may consist of a 5" overhang, 3" overhang, orblunt end.

[0094] Retroviral vectors integrate into eukaryotic genomes by adistinct mechanism of non-homologous recombination that is catalyzed bythe action of the virally encoded integrase enzyme, and the mechanism ofviral integration, replication and infection has been well described(reference 0). The mutagenic ability of retroviruses and retroviralvectors and their ability to enable the rapid identification of mutatedgenes through the linkage of retroviral tag sequences within thetranscripts of mutagenized genes are well known in the art (reference2-5).

[0095] General reference for mechanisms of retroviral infection,replication, and integration: 0: In: Retroviruses. Coffin, JM.; Hughes,SH.; Varmus, HE.Plainview (NY): Cold Spring Harbor Laboratory Press;c1997;Use of wildtype retroviruses as mutagens: 1: Varmus HE, QuintrellN, Ortiz S. Cell. 1981 Jul;25(1):23-36; Use of retrovirus promoter trapsas mutagens and to isolate trapped genes: 2: Friedrich G, Soriano P.Methods Enzymol. 1993;225:681-701; 3: Gossler A, Joyner AL, Rossant J,Skarnes WC. Science. 1989 Apr 28;244(4903):463-5; 4: Friedrich G,Soriano P. Genes Dev. 1991 Sep;5(9):1513-23; 5: von Melchner H,DeGregori JV, Rayburn H, Reddy S, Friedel C, Ruley HE. Genes Dev. 1992Jun;6(6):919-27; Randomness of retroviral insertion: 6: King W, PatelMD, Lobel LI, Goff SP, Nguyen-Huu MC. Science. 1985 May3;228(4699):554-8; 7: Hubbard SC, Walls L, Ruley HE, Muchmore EA. J BiolChem. 1994 Feb 4;269(5):3717-24.

[0096] Like retroviruses, transposons and transposon vectors can also beused to integrate sequences that an act as insertional mutagens. Alsolike retroviruses, transposons integrate by enzymatically catalyzednon-homologous recombination in which transposase enzymes catalyze thegenomic integration and transposition of transposon DNA (reference 1, 2,12, 13). Numerous transposons have been characterized that function ininsects (reference 13-15), plants (reference 16-20) and vertebrates(including mammals, reference 3-12). In particular, the TC1/marinerderivative transposon, Sleeping Beauty, has been demonstrated tointegrate efficiently in mammals. Transposons have been shown tofunction as efficient insertional mutagens in numerous systems(reference 5, 15, 17, 24-26), and to exhibit broad target specificity(reference 21-23). Transposase catalyzes SB transposition andintegration: 1: Cui Z, Geurts AM, Liu G, Kaufman CD, Hackett PB. J MolBiol. 2002 May 17;318(5):1221-35; 2: Izsvak Z, Khare D, Behlke J,Heinemann U, Plasterk RH, Ivics Z. J Biol Chem. 2002 Jun 24 SBtransposon can transpose and act as an insertional mutagen in mammals:3: Dupuy AJ, Clark K, Carlson CM, Fritz S, Davidson AE, Markley KM,Finley K, Fletcher CF, Ekker SC, Hackett PB, Horn S, Largaespada DA.Proc Natl Acad Sci U S A. 2002 Apr 2;99(7):4495-9; 4: Horie K, KuroiwaA, Ikawa M, Okabe M, Kondoh G, Matsuda Y, Takeda J. Proc Natl Acad Sci US A. 2001 Jul 31;98(16):9191-6; 5: Dupuy AJ, Fritz S, Largaespada DA.Genesis. 2001 Jun;30(2):82-8; 6: Fischer SE, Wienholds E, Plasterk RH.Proc Natl Acad Sci U S A. 2001 Jun 5;98(12):6759-64; 7: Ivics Z, HackettPB, Plasterk RH, Izsvak Z. Cell. 1997 Nov 14;91(4):501-10. Othertransposons also function in mammals: 8: Zagoraiou L, Drabek D, AlexakiS, Guy JA, Klinakis AG, Langeveld A, Skavdis G, Mamalaki C, Grosveld F,Savakis C. Proc Natl Acad Sci U S A. 2001 Sep 25;98(20):11474-8; 9:Sherman A, Dawson A, Mather C, Gilhooley H, Li Y, Mitchell R, FinneganD, Sang H. Nat Biotechnol. 1998 Nov;16(11):1050-3; 10: Kawakami K, ShimaA, Kawakami N. Proc Natl Acad Sci U S A. 2000 Oct 10;97(21):11403-8; 11:Fadool JM, Hartl DL, Dowling JE. Proc Natl Acad Sci U S A. 1998 Apr28;95(9):5182-6; 12: Plasterk RH. Cell. 1993 Sep 10;74(5):781-6. Pelements developed as insertional mutagen in invertebrates: 13: KaufmanPD, Rio DC. Nucleic Acids Res. 1991 Nov 25;19(22):6336; 14: Rubin GM,Spradling AC. Nucleic Acids Res. 1983 Sep 24;11(18):6341-51; 15:Spradling AC, Rubin GM. Science. 1982 Oct 22;218(4570):341-7. Ac and Dsand other plant transposons transpose, integrate and are used asinsertional mutagens in plants: 16: Grevelding C, Becker D, Kunze R, vonMenges A, Fantes V, Schell J, Masterson R. Proc Natl Acad Sci U S A.1992 Jul 1;89(13):6085-9; 17: Walbot V. Curr Opin Plant Biol. 2000Apr;3(2):103-7; 18: Pereira A, Aarts MG. Methods Mol Biol.1998;82:329-38; 19: Cooley MB, Goldsbrough AP, Still DW, Yoder JI. MolGen Genet. 1996 Aug 27;252(1-2):184-94; 20: Bhatt AM, Page T, Lawson EJ,Lister C, Dean C. Plant J. 1996 Jun;9(6):935-45. P element transposoncan integrate broadly throughout genomes: 21: Kassis JA, Noll E,VanSickle EP, Odenwald WF, Perrimon N. Proc Natl Acad Sci U S A. 1992Mar 1;89(5):1919-23; 22: Berg CA, Spradling AC. Genetics. 1991Mar;127(3):515-24; 23: Tower J, Karpen GH, Craig N, Spradling AC.Genetics. 1993 Feb;133(2):347-59; 24: Cooley L, Berg C, Kelley R,McKearin D, Spradling A. Prog Nucleic Acid Res Mol Biol. 1989;36:99-109;25: Cooley L, Kelley R, Spradling A. Science. 1988 Mar4;239(4844):1121-8; 26: Spradling AC, Stern DM, Kiss I, Roote J, LavertyT, Rubin GM. Proc Natl Acad Sci U S A. 1995 Nov 21;92(24):10824-30.

[0097] As used herein, the term phenotype means any property of a cellor organism. A phenotype can simply be a change in expression of an mRNAor protein. Examples of phenotypes also include, but are in no waylimited to, cellular, biochemical, histological, behavioral, or wholeorganismal properties that can be detected by the artisan. Phenotypesinclude, but are not limited to, cellular transformation, cellmigration, cell morphology, cell activation, resistance or sensitivityto drugs or chemicals, resistance or sensitivity to pathogenic proteinlocalization within the cell (e.g. translocation of a protein from thecytoplasm to the nucleus), profile of secreted or cell surface proteins,(e.g., bacterial or viral) infection, post-translational modifications,protein localization within the cell (e.g. translocation of a proteinfrom the cytoplasm to the nucleus), profile of secreted or cell surfaceproteins, cell proliferation, signal transduction, metabolic defects orenhancements, transcriptional activity, cell or organ transcriptprofiles (e.g., as detected using gene chips), apoptosis resistance orsensitivity, animal behavior, organ histology, blood chemistry,biochemical activities, gross morphological properties, life span, tumorsusceptibility, weight, height/length, immune function, organ function,any disease state, and other properties known in the art. In certainsituations and therefore in certain embodiments of the invention, theeffects of mutation of one or more genes in a cell or organism can bedetermined by observing a change in one or more given phenotypes (e.g.,in one or more given structural or functional features such as one ormore of the phenotypes indicated above) of the mutated cell or organismcompared to the same structural or functional feature(s) in acorresponding wild-type or (non-mutated) cell or organism (e.g., a cellor organism that in which the gene(s) have not been mutated).

[0098] As used herein, the term multiploid means any ploidy greater thanhaploid. Multiploid encompasses diploid, triploid, tetraploid, andaneuploid.

[0099] As used herein library means more than one cell. A library may becells subjected to one or both mutagenesis methods, singly or more thanone time. Thus a library includes, but is not limited to, one or moreclones of mutated cells or mutated cells where each cell has a differentset of mutations. Libraries provide a source of cells to subject tomutagenesis and a source of cells to screen for desired phenotypesfollowing mutagenesis.

[0100] A known gene is directed to the level of characterization of agene. The invention allows expression of genes that have beencharacterized, as well as expression of genes that have not beencharacterized. Different levels of characterization are possible. Theseinclude detailed characterization, such as cloning, DNA, RNA, and/orprotein sequencing, and relating the regulation and function of the geneto the cloned sequence (e.g., recognition of promoter and enhancersequences, functions of the open reading frames, introns, and the like).Characterization can be less detailed, such as having mapped a gene andrelated function, or having a partial amino acid or nucleotide sequence,or having purified a protein and ascertained a function.Characterization may be minimal, as when a nucleotide or amino acidsequence is known or a protein has been isolated but the function isunknown. Alternatively, a function may be known but the associatedprotein or nucleotide sequence is not known or is known but has not beencorrelated to the function. Finally, there may be no characterization inthat both the existence of the gene and its function are not known. Theinvention allows expression of any gene at any of these or otherspecific degrees of characterization.

[0101] The invention could also be practiced with a combination ofphysicochemical or insertional mutation with epigenetic techniques formodifying genome function, such as modification of methylation, is RNAantisense RNA, ribozymes, transcription factors designed to activate orinactivate multiple transcriptional regulatory sequences, modifiers ofchromosomal proteins, such as histones, that modify the transcriptionalavailability of multiple genes.

[0102]OverviewThe ability to create tagged mutations in multiple genesin multiploid cells and multicellular organisms would have utility inmany areas, including correlating a phenotype with the genes responsiblefor it by gene identification, gene discovery, determining genefunction, creating phenotypes, discovering drug targets, and makinghuman disease models in cells and in multicellular organisms.

[0103] The ability to create tagged homozygous mutations in a cell ormulticellular organism enables alteration of the genetic make up of acell and has numerous uses, such as those above as well as correctinggenetic defects. The in vitro, ex vivo, and in vivo potential uses ofthis technology are enormous and will be readily apparent to the skilledartisan.

[0104] The present invention, therefore, is directed to methods formutating a single gene or multiple genes (e.g., two or more) ineukaryotic cells and multicellular organisms. The invention also isdirected to insertional mutagens for making the mutant cells andorganisms, and which also can be used to analyze the mutations that aremade in the cells and organisms. The invention also is directed tomethods in which one or more mutated genes is tagged by a tag providedby the insertional mutagen to allow the detection, selection, isolation,and manipulation of a cell with a genome tagged by the insertionalmutagen and allows the identification and isolation of the mutatedgene(s).

[0105] The invention provides methods for making multiple mutations(i.e., mutations in two or more genes that produce a phenotypecumulatively) in cells and organisms and tagging at least one of themutated genes such that it can be rapidly recovered and identified.Creation of multiple mutations in a cell where at least one of themutations is tagged is useful in studying gene function. One reason forthis is that many phenotypes require multiple gene mutations in order tobe manifested. Current methods do not allow for creation of multiplemutations in a cell in a manner that allows easy identification of themutated genes. The present invention enables multiple mutations to becreated in the same cell and allows at least one of the mutations to betagged.

[0106] Libraries that contain the cells mutagenized by a combination ofinsertional mutagenesis and physicochemical mutagenesis can be screenedfor a phenotype of interest. In cells that have the phenotype ofinterest, one or more tagged genes can be identified and validated asbeing responsible for the particular phenotype of interest.

[0107] The invention also provides methods for making homozygousmutations in eukaryotic cells and organisms. The homozygously mutatedgene is tagged by an insertional mutagen so that it can be identifiedand, if desired, recovered. Homozygous mutations are useful fordiscovering functions associated with the mutated gene.

[0108] The methods of the present invention can be used to mutate anyeukaryotic cell, including, but not limited to, haploid (in the case ofmultiple gene mutations), diploid, triploid, tetraploid, or aneuploid.In one embodiment, the cell is diploid. Cells in which the methods ofthe present invention can be advantageously used include, but are notlimited to, primary cells (e.g., cells that have been explanted directlyfrom a donor organism) or secondary cells (e.g., primary cells that havebeen grown and that have divided for some period of time in vitro, e.g.,for 10-100 generations). Such primary or secondary cells can be derivedfrom multi-cellular organisms, or single-celled organisms. The cellsused in accordance with the invention include normal cells, terminallydifferentiated cells, or immortalized cells (including cell lines, whichcan be normal, established or transformed), and can be differentiated(e.g., somatic cells or germ cells) or undifferentiated (e.g.,multipotent, pluripotent or totipotent stem cells).

[0109] Examples of tissues from which cells can be isolated for use inthe present invention include, without limitation, neuronal tissue(including tissue from the central and peripheral nervous systems),hematopoietic tissue, lymphatic tissue, immune tissue, bone tissue,stromal tissue (including, e.g., bone marrow tissue), mesenchymaltissue, mesothelial tissue, connective tissue (including e.g.,cartilage, dermal tissue, subcutaneous tissue, adipose tissue, etc.),endothelial tissue, epithelial tissue, lung tissue, skin tissue, kidneytissue, gastrointestinal tissue (including esophagus, stomach,intestine, etc.), brain tissue, heart tissue, pancreatic tissue, muscletissue, liver tissue, gonadal tissue, embryonic tissue includingembryonic stem cells and embryonic germ cells), zygote tissue,embryonic, and other cells and tissue known in the art.

[0110] A variety of cells isolated from the above-referenced tissues, orobtained from other sources (e.g., commercial sources or cell banks),can be used in accordance with the invention. Non-limiting examples ofsuch cells include somatic cells such as blood cells (erythrocytes andleukocytes), endothelial cells, epithelial cells, neuronal cells (fromthe central or peripheral nervous systems), muscle cells (includingmyocytes and myoblasts from skeletal, smooth or cardiac muscle),connective tissue cells (including fibroblasts, adipocytes,chondrocytes, chondroblasts, osteocytes and osteoblasts) and otherstromal cells (e.g., macrophages, dendritic cells, thymic nurse cells,Schwann cells, etc.). Eukaryotic germ cells (spermatocytes and oocytes)can also be used in accordance with the invention, as can theprogenitors, precursors and stem cells that give rise to theabove-described somatic and germ cells. These cells, tissues and organscan be normal, or they can be pathological such as those involved indiseases or physical disorders, including but not limited to infectiousdiseases (caused by bacteria, fungi or yeast, viruses (including HIV) orparasites), in genetic or biochemical pathologies (e.g., cysticfibrosis, hemophilia, Alzheimer's disease, schizophrenia, musculardystrophy, multiple sclerosis, etc.), or in carcinogenesis and othercancer-related processes.

[0111] The eukaryotic cells used in the methods of the present inventioncan be animal cells, plant cells (monocot or dicot plants) or fungalcells, such as yeast. Animal cells include those of vertebrate orinvertebrate origin. Vertebrate cells are of particular use in thepresent invention, especially mammalian cells (including, but notlimited to, cells obtained or derived from human, simian or othernon-human primate, mouse, rat, avian, bovine, porcine, ovine, canine,feline and the like), avian cells, fish cells (including zebrafishcells), insect cells (including, but not limited to, cells obtained orderived from Drosophila species, from Spodoptera species (e.g., Sf9obtained or derived from S. frugiperida, or HIGH FIVE™cells) or fromTrichoplusa species (e.g., MG1, derived from T. ni)), worm cells (e.g.,those obtained or derived from C. elegans), and the like. It will beappreciated by the ordinarily skilled artisan, however, that cells fromany species besides those specifically disclosed herein can beadvantageously used in accordance with the methods of the presentinvention, using art-known methods in conjunction with those describedherein and without the need for undue experimentation.

[0112] Cell lines are also useful in the present invention. Examples ofuseful cell lines include, but are not limited to, HT1080 cells (ATCCCCL 121), HeLa cells and derivatives of HeLa cells (ATCC CCL 2, 2.1 and2.2), MCF-7 breast cancer cells (ATCC BTH 22), K-562 leukemia cells(ATCC CCL 243), KB carcinoma cells (ATCC CCL 17), 2780AD ovariancarcinoma cells (see Van der Blick, A. M. et al., Cancer Res.48:5927-5932 (1988), Raji cells (ATCC CCL 86), Jurkat cells (ATCC TIB152), Namalwa cells (ATCC CRL 1432), HL-60 cells (ATCC CCL 240), Daudicells (ATCC CCL 213), RPMI 8226 cells (ATCC CCL 155), U-937 cells (ATCCCRL 1593), Bowes Melanoma cells (ATCC CRL 9607), WI-38VA13 subline 2R4cells (ATCC CLL 75.1), and MOLT-4 cells (ATCC CRL 1582), as well asheterohybridoma cells produced by fusion of human cells and cells ofanother species. Secondary human fibroblast strains, such as WI-38 (ATCCCCL 75) and MRC-5 (ATCC CCL 171) can also be used. Other mammalian cellsand cell lines can be used in accordance with the present invention,including but not limited to CHO cells, COS cells, VERO cells, 293cells, PER-C6 cells, M1 cells, NS-1 cells, COS-7 cells, MDBK cells, MDCKcells, MRC-5 cells, WI-38 cells, WEHI cells, SP2/0 cells, BHK cells(including BHK-21 cells); these and other cells and cell lines areavailable commercially, for example from the American Type CultureCollection (P.O. Box 1549, Manassas, Virginia 20108 USA). Many othercell lines are known in the art and will be familiar to the ordinarilyskilled artisan; such cell lines therefore can be used equally well inthe methods of the present invention.

[0113] The present invention can be practiced using plant cells. Methodsfor culturing plant cells, insertionally mutating plant cells, andproducing transgenic plants are known in the art (see, e.g., Hall,Robert D., Plant Cell Culture Protocols, Humana Press, New Jersey(1999); Gartland and Davey, Agrobacterium Protocols, Humana Press, NewJersey (1995); each incorporated herein by reference for teachingmethods of culturing, transfecting, mutating, and producing transgenicplants and plant cells).

[0114] In certain embodiments of the invention, cells can be mutatedwithin the organism or within the native environment as in tissueexplants (e.g., in vivo or in situ). Alternatively, tissues or cellsisolated from the organism using art-known methods and genes can bemutated according to the present methods. The tissues or cells areeither maintained in culture (e.g., in vitro), or re-implanted into atissue or organism (e.g., ex vivo).

[0115]Methods of Making Mutant CellsThe invention involves two types ofmutagenesis events physicochemical and insertional. In one aspect,multiple genes are mutated which cumulatively produce a desiredphenotype. The mutations can be homozygous, heterozygous, or acombination of the two. One or more of these genes is tagged by means ofan insertional mutagen. A single cell can contain all of the taggedgenes or less than all of the tagged genes, for example, one taggedgene. Accordingly, in a non-limiting example two mutated genes in thesame cell are required to produce the desired phenotype. Cells aremutated to produce the phenotype, screened for the desired phenotype andtwo cells are selected that have the phenotype because both genes aremutated in each cell. One gene is tagged in one cell and the other geneis tagged in the second cell. This process allows the identification ofboth genes that cumulatively produce the desired phenotype even thoughonly one gene was tagged in each cell.

[0116] Furthermore, the present invention is useful even if only onegene responsible for a phenotype is identified. Such a gene could form atarget for modulating the phenotype. This is useful, for example, indrug discovery and in production of useful gene products. Accordingly,one or more of multiple genes required to produce a desired phenotypecan be identified using the mutagenic methods of the invention.

[0117] The invention also involves using the mutagenic methods toproduce a desired phenotype that results from the homozygous mutation ofa single gene. At least one of the mutated alleles is tagged.

[0118] The mutation process involves at least one physicochemicalmutagenic event, and at least one insertional mutagenic event, which canbe carried out in any order or simultaneously.

[0119]Physicochemical MutagenesisOne phase of the mutational methods ofthe present invention involves physicochemical mutagenesis of a cell ororganism by one or more physicochemical mutagens. In one embodiment,physicochemical mutagenesis is the initial event in the mutagenicprocess. In another embodiment, it can be performed simultaneously withan insertional mutagenesis event. In another embodiment it follows aninitial insertional mutagenesis event. In any of these embodiments oneor more further physiochemical and/or insertional mutagenesis events canbe carried out in any order and simultaneously.

[0120] Physicochemical mutagenesis can be achieved by a variety ofmutagenic agents. Examples of mutagenic agents known in the art include,but are not limited to, chemical mutagens (e.g., DNA-intercalating orDNA-binding chemicals which affect (e.g., increase or decrease) theactivity, protein coding potential or expression of a gene contained ona DNA molecule to which the chemical has bound), physical mutagens(e.g., UV light, ionizing radiation, (gamma, beta and alpha radiation,x-rays), biochemical mutagens (e.g., restriction enzymes, DNA repairmutagens, DNA repair inhibitors, and error-prone DNA polymerases andreplication proteins), and the like. The mutagenic changes in DNAsequence can occur as a direct consequence of the mutagen/DNAinteraction. Alternatively DNA repair mechanisms induced in response todamage inflicted by the mutagen may participate in implementingmutations.

[0121] In certain embodiments, chemical mutagenesis is used to inducemutation in one or more genes in the target cell or organism. An exampleof a chemical mutagen commonly used to mutate cells and organisms isN-ethyl-N-nitrosourea (ENU). Other examples of chemical mutagens usefulin the present invention include, but are not limited to,ethylmethanesulphonate (EMS) and ICR191. Many other chemical mutagensare known in the art and are useful in the present invention (see, e.g.,E. C. Friedberg, G. C. Walker, W. Siede, DNA Repair and Mutagenesis, ASMPress, Washington DC (1995); 1: Justice MJ, Zheng B, Woychik RP, BradleyA. Methods. 1997 Dec;13(4):423-36; 2: Gasparro FP, Felli A, Schmitt IM.Recent Results Cancer Res. 1997;143:101-27; 3: Vogel EW, Natarajan AT.Mutat Res. 1995 Aug;330(1-2):183-208; 4: Anderson P. Methods Cell Biol.1995;48:31-58; 5: Russell LB. Environ Mol Mutagen. 1994;23 Suppl24:23-9; 6: DeMarini DM, Brockman HE, de Serres FJ, Evans HH, StankowskiLF Jr, Hsie AW. Mutat Res. 1989 Jan;220(1):11-29; 7: Wood RD, SedgwickSG. Mutagenesis. 1986 Nov;1(6):399-405; 8: Loprieno N, Barale R, VonHalle ES, von Borstel RC. Mutat Res. 1983 Jun;115(2):215-23; 9: BradleyMO, Bhuyan B, Francis MC, Langenbach R, Peterson A, Huberman E. MutatRes. 1981 Sep;87(2):81-142; 10: Hoffmann GR. Mutat Res. 1980Jan;75(1):63-129; 1: Flessel CP. Adv Exp Med Biol. 1977;91:117-28; 12:McCann J, Ames BN. Proc Natl Acad Sci U S A. 1976 Mar;73(3):950-4; 13:Orias E, et. al. Methods Cell Biol. 1976;13:247-82; 14: Kilbey BJ.Methods Cell Biol. 1975;12:209-31 incorporated herein by reference forteaching chemical mutagens and their use in inducing gene mutation invarious cells and organisms).

[0122] Preferred doses of chemical mutagens for inducing mutations incells and organisms are described elsewhere herein, are known in theart, or can be readily determined by the ordinarily skilled artisanusing assays of mutagenesis described herein or known in the art.Chemical mutagenesis of cells in vitro can be achieved by treating thecells with various doses of the mutagenic agent and/or controlling thetime of exposure to the agent. By titrating the mutagenic agent exposureand/or dose, it is possible to carry out the optimal degree ofmutagenesis for the intended purpose, thereby mutating a desired numberof genes in each cell. Examples of useful doses of ENU are 0.1 - 0.4mg/ml for 1 - 2 hours. Examples of useful doses of EMS are 0.1 - 1 mg/mlfor 10 - 30 hours. While the treatment conditions described herein areknown to be useful in accordance with the present invention, lower andhigher doses and exposure times can be used to achieve the desiredmutation frequency. In addition, optimal doses and exposure times willvary from cell type to cell type and can be readily determined using themethods of the invention, knowledge that is readily available in theart, and routine experimentation. Other chemical mutagens and mutagenicagents are used at doses known in the art or determined through routineexperimentation.

[0123] Physicochemical mutagenesis can be carried out using a singlemutagenic agent. Alternatively, physicochemical mutagenesis can becarried out using multiple (more than one) mutagenic agents. Whenmultiple mutagenic agents are used, mutagenesis can be carried out byexposing the cells to the agents at the same time or sequentially.Physicochemical mutagenesis of a given cell or organism can also becarried out in a single exposure or in multiple sequential exposures, inany order.

[0124] If physicochemical mutagenesis of a cell or organism is performedprior to another mutagenic event (e.g., insertional mutagenesis oranother round of physicochemical mutagenesis) carried out on the samecell or organism, the mutation frequency in the physicochemicallymutated cell or organism optionally can be determined prior to orfollowing the additional mutagenic event (e.g., prior to or followinginsertional mutagenesis or the next round of physicochemicalmutagenesis). This approach allows the artisan to select the mutagenicagent and conditions that achieve the desired mutation frequency.

[0125] Mutation frequency obtained with a particular mutagen can beassessed using a number of methods described herein and known in theart. One such method is to test for gene function of a selectable orscreenable marker within the cell. Typically, the marker used in thisapproach exists in the cell as a single copy gene; however, multi-copygenes could be useful in such methods, particularly to assess highmutation frequencies. The marker gene can be a cellular gene, such asHPRT, or could be an exogenously introduced gene, such as HSV-thymidinekinase, green fluorescent protein, luciferase, βa cell surface proteinsuitable for FACS sorting, and the like. Following mutagenesis, theartisan can assay for the absence of, or reduction in, marker geneexpression or activity. For example, mutation frequency can be assessedin mutagenized cells by selecting against HPRT (endogenous HPRT is Xandtherefore, there is normally only one functional copy in a given cell)or HSV-TK (in cells transfected with a single copy of the HSV-TK gene)using selection with 6-thioguanine (6-TG) and1,2'-deoxy-2'-fluoro-o-D-arabinofuranosyl-5-iodouracil (FAIU),respectively. The number of surviving clones divided by the total numberof clones plated then defines the mutation frequency for single copygenes. Thus, a desired mutation frequency can be obtained in any cell ororganism by mutagenizing cells under several conditions, assaying formarker activity, and determining mutation frequency. With respect to theother marker examples described above, and those known in the art, anysuitable assay can be used to test for loss of marker function (or gainof function) including enzyme assays, ELISA, and FACS. Once a desiredlevel of mutation in a cell population is obtained, one or more cellsfrom that population can be used in the second phase of the process, asdescribed below.

[0126] Another method for assessing mutation frequency is to amplify(e.g., via PCR) a genomic locus or cDNA from one cell (or a populationof cells) and clone the amplification product(s), and sequence thecloned nucleotides. By comparing the sequence from the mutagenized cellto the sequence from the wild-type (non-mutagenized) cell, it ispossible to determine the number of mutations per unit length, (e.g.,one mutation per 1000 bp). A variety of permutations of this method, andsimilar methods, will be readily apparent to a person of skill in theart. Thus, this assay can be used to select mutagenesis conditions thatgive rise to a desired mutation frequency.

[0127] The present invention involves mutagenizing cells withphysicochemical mutagens, such as those mutagens that are well-known inthe art and that therefore will be familiar to the ordinarily skilledartisan, such that one or more genes are mutagenized in each cell.

[0128] Any number of genes can be mutagenized in each cell. To select amutation frequency and library coverage, it is useful to consider thatthere is a relationship between the total number of genes contained inthe genome of the cell, the number of mutated genes produced in eachcell, the number of mutated cells generated, and the complexity of thelibrary. In general, as the number of genes in a cell increases (e.g.yeast versus mammalian cells), the number of mutations per cell or thenumber of mutated cells (or both) must increase in order to produce thesame library coverage.

[0129] As a general guideline, creation of a larger number of mutationsper cell reduces the number of cells required to produce a library ofcells in which every gene in the genome has been mutated, on average, atleast one time. Likewise, as the mutation frequency increases, the sizeof the library can decrease without affecting its coverage. For example,if one copy of a given gene is mutated at a frequency of approximately 1cell out of 100 surviving mutagenized cells, then 100 mutant cell cloneswill be necessary to produce a library in which one copy of each genehas been mutated, on average, one time. However, if one copy of a geneis mutated at a frequency of 1 cell in 30 cells, then only 30 mutantclones are necessary to produce the same IX library. A library of thissize can be referred as a 1X library to indicate that one copy of eachgene has been mutated one time, on average.

[0130] In preferred embodiments, at least 50-10,000 (one mutated copy of0.05-10% of the total number of alleles in the diploid genome) genes aremutated in each cell. In highly preferred embodiments, at least250-5,000(one mutated copy of 0.25-5% of the total number of alleles inthe diploid genome) genes are mutated in each cell. In otherembodiments, it is useful to mutate 10-100 genes in each cell. In stillother embodiments, it is useful to mutate 10,000-50,000 genes, or more.As the number of mutations per cell increases it becomes increasinglylikely that both alleles of some genes will be mutated as well asbecoming increasingly likely that multiple genes that are needed for aparticular biological function will be mutated in a single cell. Thusmutagenizing a cell can sensitize this cell or libraries of similarlymutagenized cells to the induction of phenotypes as a consequence offurther mutation (e.g. insertional mutagenesis) on this sensitizedgenetic background.

[0131] It should be noted that a 1X library is only an estimate oflibrary complexity. Statistically, assuming a random mutation frequency,a 1X library will contain about 66% of the genes with a single mutation.Some genes may be mutated twice (where the genome in the cell isdiploid) and others will not be mutated. To increase the probabilitythat most or all genes in a given library are mutated at least once, alibrary of increased complexity can be used (e.g., a 5X or 10X librarycan be used). In other embodiments that do not require mutation of everygene in a genome, libraries of less complexity can be created (e.g., a0.1X (or less) library).

[0132] It is also possible to estimate library size and coverage byassessing the mutation frequency of individual genes in the library. Forexample, if HPRT and HSV-TK are both mutated at a frequency of 1 in 500cells under a specific mutation condition, then a 1X library could beestimated to contain 500 cells. Such libraries of increased or decreasedcomplexity can be created by manipulating the conditions ofphysicochemical mutagenesis to which the cells are exposed, e.g., byincreasing the dose and/or exposure time of the physicochemicalmutagen(s) to make libraries of increased complexity, or by decreasingthe dose and/or exposure time of the physicochemical mutagen(s) forpreparation of libraries of decreased complexity.

[0133] If physicochemical mutagenesis is carried out prior toinsertional mutagenesis, the physicochemically mutated cells or organismoptionally can be screened at this time for a desired phenotype. Thisscreening can be done whether or not the mutation frequency in thephysicochemically mutated cells is determined. Mutant cells that displaythe desired phenotype prior to insertional mutagenesis can then bediscarded; it is desirable to remove cells displaying the desiredphenotype at this stage since they would interfere with subsequentscreening by displaying the desired phenotype independent of insertionalmutagenesis. As disclosed herein, insertional mutagenesis provides a wayto tag a gene that is involved in the induction of a desired phenotype.If the phenotype is already produced by the physicochemical mutagenesisof a gene, then an insertional tag cannot be used to correlate thetagged gene with the phenotype since the phenotype is not induced by theinsertion event. Thus, in one embodiment of the present invention, cellsare assayed for desired phenotypes prior to insertional mutation toidentify and remove cells that have acquired the phenotype withoutinsertional mutagenesis.

[0134] In another embodiment of the invention where physicochemicalmutagenesis is carried out prior to insertional mutagenesis, phenotypesare not assessed prior to insertional mutation because it is unlikelythat the desired phenotype will be produced. In this embodiment, it ispreferable to mutate cells under conditions that yield a low number ofmutations per cell because it is unlikely that the desired phenotypewill be produced. The reason for this is that when a large number ofmutations are made in each cell, for example 10,000 mutated genes percell, the probability of mutating two genes or alleles to create aphenotype is relatively high. Conversely, when a small number ofmutations is made in each cell, for example 100 mutated genes per cell,the probability of mutating two or more required genes or alleles in thesame cell to create a phenotype is low. Furthermore, the number ofphysicochemically mutated cells selected for insertional mutagenesisalso impacts the probability that a cell will be selected that has thephenotype prior to insertional mutagenesis. For example, if 1,000mutations are created in each cell by physicochemical mutagenesis, butonly one cell is selected for insertional mutagenesis, the probabilitythat this one cell possesses the desired phenotype can be relativelylow. On the other hand, if 10 gene mutations are created in each cell byphysicomutagenesis, but 10⁶ physicochemically mutated clones areselected for insertional mutagenesis, then the probability that one ormore cells possess the desired phenotype prior to insertionalmutagenesis can be relatively high. Thus, there is a correlation betweenthe number of physicochemical mutations per cell, the number ofphysicochemically mutated cells selected for insertional mutagenesis,and the probability of selecting one or more cells displaying aphenotype prior to insertional mutagenesis. One method for empiricallyassessing this relationship for a given phenotype is tophysicochemically mutate cells under several conditions and then testfor the phenotype frequency prior to insertional mutagenesis. If, forexample, the phenotype frequency for a particular physicochemicalmutation is found to be 1 mutant phenotype observed per 1000 cells, byselecting 50 cells there is only approximately a 5% probability ofselecting a cell displaying the phenotype of interest. Another approachfor assessing the likelihood of selecting a physicochemically mutatedcell displaying the desired phenotype is to determine the mutationfrequency in a given population of mutated cells using one of the assaysdescribed above (e.g., using HPRT or HSV-TK selection assays). If, forexample, the mutation frequency for a single copy gene is 1 mutation in400 clones, then the probability of mutating both alleles of a gene in adiploid cell is roughly 1 in 160,000 (1/400 x 1/400). By selecting 4000cells for insertional mutagenesis, the probability of cells displaying aphenotype caused by a homozygous gene mutation is, therefore, relativelylow.

[0135] The foregoing assays for determining mutation frequency areintended as nonexamples provided solely to impart guidelines to theartisan for selecting cells for insertional mutagenesis. As theordinarily skilled artisan will be aware, there are a number of otherassays of mutation frequency that can be employed in conjunction withthe present invention; the exemplary assays described herein thereforeshould not be construed as limiting the scope of the invention in anyway.

[0136]Insertional MutagenesisThe other mutational method of the presentinvention is insertional mutagenesis of a cell or organism by one ormore insertional mutagens (which can be the same or different). In oneembodiment, insertional mutagenesis is the initial mutagenic event andprecedes a physicochemical mutagenic event. In another embodiment,insertional mutagenesis is carried out simultaneously withphysicochemical mutagenesis. In another embodiment, an insertionalmutagenesis event follows a physicochemical mutagenesis event. In any ofthese embodiments, the cells can subsequently be subjected to one ormore additional physicochemical mutagenesis events, one or moreadditional insertional mutagenesis events, or a combination of anynumber of these events in any order or simultaneously. The coupling ofinsertional mutagenesis with one or more additional mutational eventsprovides the ability to create mutations, in cells or organisms, thathave previously been difficult or impossible to produce or to detect.

[0137] According to the invention, insertional mutagenesis involves theintegration of one or more polynucleotide sequences into the genome of acell or organism to mutate one or more endogenous genes in the cell ororganism. Thus, the insertional mutagenic polynucleotides of the presentinvention are designed to mutate one or more endogenous genes when thepolynucleotides integrate into the genome of the cell.

[0138]Insertional MutagensAccordingly, the insertional mutagens used inthe present invention can comprise any nucleotide sequence capable ofaltering gene expression levels or activity of a gene product uponinsertion into DNA that contains the gene. The insertional mutagens canbe any polynucleotide, including DNA and RNA, or hybrids of DNA and RNA,and can be single-stranded or double-stranded, naturally occurring ornon-naturally occurring (e.g., phosphorothioate, peptide-nucleic acids,etc.). The insertional mutagens can be of any geometry, including butnot limited to linear, circular, coiled, supercoiled, branched, hairpin,and the like, and can be any length capable of facilitating mutation,and tagging of an endogenous gene. Typically, the insertional mutagensare at least 5 nucleotides in length, at least 10 nucleotides in length,at least 15 nucleotides in length, at least 20 nucleotides in length, atleast 25 nucleotides in length, at least 50 nucleotides in length, atleast 100 nucleotides in length, at least 200 nucleotides in length, atleast 250 nucleotides in length, at least 500 nucleotides in length, atleast 1000 nucleotides (e.g., at least 1 kb) in length, etc. In someembodiments of the invention, the insertional mutagens can be at least 1kb in length, at least 2 kb in length, at least 2.5 kb in length, atleast 5 kb in length, at least 7.5 kb in length, at least 10 kb inlength, or larger. Preferably, the insertional mutagens at least 10-15nucleotides in length. This length allows the artisan to use nucleotideprimers complementary to the inserted polynucleotide to be used to makeprimer extension products of the insertionally mutated gene to detect orcharacterize (e.g., sequence) the gene.

[0139] In certain embodiments of the invention, the insertional mutagenscan comprise one or more nucleotide sequences that provide a desiredfunction. Such nucleotides sequences include, but not are not limitedto, one or more multiple cloning sites, one or more transcriptiontermination sites, one or more transcriptional regulatory sequences(e.g., one or more promoters, enhancers, or repressors), one or moresequences that encode translational signals, one or more open readingframes (ORFs), one or more sequences mutating ORFs, one or more stopcodons, one or more sequences mutating or eliminating stop codons, oneor more mRNA destabilizing elements, one or more RNA stabilizingelements, one or more sequences that result in the formation of hairpinloops, one or more sequences that disrupt or eliminate hairpin loops,one or more reporter genes, one or more splice acceptor sequences, oneor more splice donor sequences, one or more internal ribosome entrysites (IRES), one or more transposon sequences, one or moresite-specific recombination site sequences, one or more restrictionenzyme sequences, one or more nucleotide sequences encoding a fusionpartner protein or peptide (e.g., glutathione-S-transferase (GST),hexahistidine (His₆) or thioredoxin), one or more selectable markers orselection modules, one or more bacterial sequences useful forpropagating the insertional mutagenic polynucleotide molecules in a hostcell, one or more 3' gene trap cassettes, one or more nucleotidesequences encoding localization signals such as nuclear localizationsignals or secretion signals, one or more nucleotide sequences encodingone or more transmembrane regions (e.g., one or more amino acids, andtypically one or more hydrophobic amino acids, capable of anchoring apolypeptide into a cellular membrane), one or more origins ofreplication, one or more protease cleavage sites, one or more desiredproteins or peptides encoded by a gene or a portion of a gene, a 5" genetraps on an insertional mutagen, a 3" gene trap on an insertionalmutagen, one or more selectable markers, one or more sequences encodingone or more 5' or 3' polynucleotide tails (particularly a poly (A)tail), and the like. As the ordinarily skilled artisan will readilyunderstand, the insertional mutagens of the invention can comprise oneor more of these or other nucleotide sequences, in any order andcombination, and can comprise more than one of a given nucleotidesequence.

[0140] Preferably, the insertional mutagens comprises a selectablemarker, a 5" gene trap, a splice acceptor with no operatively linkedpromoter, stop codons in all three reading frames, an IRES, a transposonsequence, or a site specific recombination site.

[0141] In one specific embodiment, the insertional mutagen comprises asplice acceptor sequence that does not contain an operably-linkedpromoter 5′ to the splice acceptor. This polynucleotide can serve as anessential 5′ gene trap. In certain other specific embodiments, theinsertional mutagen is found on a retroviral vector. The retrovirus canbe infectious or non-infectious but in a preferred embodiment theretrovirus vector can form infectious retrovirus particles. In anotherspecific embodiment, the insertional mutagen contains sequences requiredfor transposition and accordingly forms a transposable element. In afurther specific embodiment, the insertional mutagen comprises apolynucleotide having a splice acceptor with no operably-linkedpromoter, the splice acceptor having an optimal branch point, themutagen also containing three stop codons in all three reading frames,an IRES that includes an exonic splicing enhancer, the mutagen furthercomprising a selectable marker with a polyadenylation siteoperably-linked, and a polyA trap component. The polyA trap componentcan optionally be constructed as described herein and as generally knownin the art. PolyA traps are also discussed in U.S. Patent No. 6,410,266,incorporated herein by reference for disclosing polyA traps. In anotherembodiment, the insertional mutagen contains a splice acceptor withoutan operably-linked promoter 5′, the splice acceptor containing anoptimal branch point, the vector further containing three stop codons inall three reading frames, an IRES that does not contain the enhancer, aselectable marker with an operably-linked polyadenylation site, andwherein the vector does not contain the polyA trap.

[0142] In certain embodiments, the insertional mutagens comprise one ormore nucleotide sequences capable of mutating an open reading frame(ORF). For example, the insertional mutagen can contain a number ofnucleotides that is not divisible by 3, and which, therefore, wouldresult in a frame-shift upon insertion of the polynucleotide into anORF.

[0143] In another embodiment, the insertional mutagen comprises one ormore primer recognition sites, thereby facilitating the detection of themutated gene using primer-based amplification or sequencing methods suchas PCR.

[0144] In certain other embodiments of the invention, the insertionalmutagens can additionally or alternatively comprise one or more stopcodons. Stop codons are useful for terminating translation of genes,thereby facilitating mutation of a functional protein. The stop codonscan be located on one or both strands of a double stranded insertionalmutagen and can be nested to terminate translation in all three readingframes.

[0145] In certain other embodiments of the invention, the insertionalmutagens can additionally or alternatively comprise one or more mRNAdestabilizing elements. Upon integration into a gene and incorporationinto an mRNA produced by the gene, the RNA instability element willdecrease the amount of mRNA from the gene. A number of mRNA instabilityelements are known in the art and useful in the present invention (see,for example, Shaw et al., Cell 46:659-667 (1986); Ishida et al., NucleicAcids Research 27:e35 (1999) each incorporated herein by reference forteaching RNA instability elements and methods of using such elements).

[0146] In certain other embodiments of the invention, the insertionalmutagens can additionally or alternatively comprise one or moreselectable markers. A selectable marker is a gene that encodes anexpression product that can be selected for or against. Examples ofselectable markers include but are not limited to:(1) polynucleotidesegments that encode products which provide resistance against otherwisetoxic compounds (e.g., antibiotics or other drugs); (2) polynucleotidesegments that encode products which are otherwise lacking in therecipient cell (e.g., tRNA genes, auxotrophic markers); (3)polynucleotide segments that encode products which suppress the activityof a gene product; (4) polynucleotide segments that encode productswhich can be readily identified (e.g., phenotypic markers such as βgreenfluorescent protein (GFP), and cell surface proteins); (5)polynucleotide segments that bind products that are otherwisedetrimental to cell survival and/or function; (6) polynucleotidesegments that otherwise inhibit the activity of any of thepolynucleotide segments described in (1)above (e.g., antisenseoligonucleotides); (7) polynucleotide segments that bind products thatmodify a substrate (e.g., methylases and restriction endonucleases);(8)segments that can be used to isolate or identify a desired molecule(e.g. specific protein binding sites); (9) polynucleotide segments thatencode one or more screenable markers; (10) polynucleotide segments,which when absent, directly or indirectly confer resistance orsensitivity of the cell to particular compounds; (11) polynucleotidesegments that encode products which are toxic in recipient cells; (12)polynucleotide segments that inhibit replication, partition orheritability of nucleic acid molecules that contain them; and (13)polynucleotide segments that encode conditional replication functions,e.g., replication in certain hosts or host cell strains or under certainenvironmental conditions (e.g., temperature, nutritional conditions,etc.). In the present invention, selectable markers allow the detectionof integration of the insertional mutagens into the host cell genome. Inaddition, selectable markers can be positioned on the insertionalmutagens to allow selection for insertion events that occur intranscriptionally active or silent regions of the genome (see Figures2-6 for non-limiting examples). The selectable marker can be expressedfrom a promoter on the insertional mutagen that is inserted or from apromoter located in the polynucleotide to be mutated (see Figure 7 fornon-limiting examples). Selectable markers suitable for use inaccordance with this aspect of the invention include positive selectablemarkers and negative selectable markers.

[0147] A positive selectable marker allows cells expressing theselectable marker to survive selection, whereas cells not expressing theselectable marker die during selection. Examples of positive selectablemarkers include, but are not limited to, neomycin resistance gene (neo),puromycin resistance gene (puro), zeomycin resistance gene (zeo),hygromycin resistance gene (hyg), histidine D (his D), dihydro-oratase,glutamine synthetase (gs), aspartate transcarbamylase, xanthine guaninephosphoribosyl transferase (gpt), carbamyl phosphate synthase (cad),multidrug resistance 1 (mdr1), thymidine kinase (tk), and hypoxanthinephosphoribosyl transferase (HPRT). Other suitable positive selectablemarkers are known in the art and will be familiar to the ordinarilyskilled artisan. In accordance with the invention, the selectable markercan be expressed from a promoter on the insertional mutagen that isinserted, or from a promoter located in the DNA to be mutated.Accordingly, in the present invention, a positive selectable marker canbe used, for example, to select for cells that have integrated theinsertional mutagen (regardless of location in the genome) (Figure 7,selection for SM) or for cells in which the insertion is into atranscriptionally active gene (for examples, see FiguresA negativeselectable marker causes cells expressing the selectable marker to dieduring selection, whereas those cells not expressing the selectablemarker survive selection. Examples of negative selectable markersinclude but are not limited to HPRT, thymidine kinase, cholera toxin,pertussis toxin, tetanus toxin, and diphtheria toxin. Other negativeselectable markers are known in the art and will be familiar to theordinarily skilled artisan. The negative selectable marker used inaccordance with the invention can be advantageously expressed from apromoter on the insertional mutagen, or from a promoter located in theDNA to be mutated. In the present invention, a negative selectablemarker can be used, for example, to select against cells where insertionis into a transcriptionally active gene. The presence of a negativeselectable marker in combination with site-specific recombinationsignals can also be used to select against cells that have failed todelete the sequences between the recombination signals (see, e.g.,Figures 8C, 9A-9D and 9F for non-limiting examples of insertionalmutagens useful in accordance with this embodiment of the invention).

[0148] In certain such embodiments, the insertional mutagen can containone or more positive selectable markers and one or more negativeselectable markers. In such embodiments in which the insertional mutagencontains both a positive and a negative selectable marker, the markerscan be present as separate open reading frames or as a single fusionopen reading frame (see Figure 9F for a non-limiting example of such apolynucleotide). When the selectable markers are present in thepolynucleotides as separate open reading frames, the positive selectablemarker and negative selectable marker can be expressed as a singlepolycistronic transcript (see Figure 9G for a non-limiting example ofsuch a insertional mutagen) or as separate transcripts (see Figures 8Cand 9A-9C for non-limiting examples of such insertional mutagens). Thepresence of both a positive selectable marker and a negative selectablemarker in the same insertional mutagen can be used, for example, toselect against actively expressed genes and for developmentallyregulated genes, and vice versa. Alternatively, the presence of both apositive selectable marker and a negative selectable marker can be usedto select for actively expressed genes that are down-regulated inresponse to developmental or environmental cues. Vectors and methods fortrapping and selecting for developmentally regulated genes arewell-known in the art (see, e.g., Gogos et al. , J. Virol. 71:1644-1650(1997); Wempe et al., Genome Biol. 2:research 23.1-23.10 (2001); andMedico et al., Nature Biotech. 19:579-582 (2001); the disclosures of allof which are incorporated herein by reference in their entireties forthese vectors and methods).

[0149] In certain other embodiments of the invention, the insertionalmutagens can additionally or alternatively comprise one or morerecombination sites, for example one or more site-specific recombinationsites or signals (see Figures 8 and 9). These recombination sites arediscrete segments on the nucleic acid molecules that are recognized andbound by certain recombination proteins during the initial stages ofintegration or recombination between two nucleic acid molecules thateach comprise such a recombination site. As discussed in detail above,such site-specific recombination sites or signals are useful fordeleting (or inverting) the inserted insertional mutagen or a portionthereof from the DNA into which the insertional mutagen as inserted.This is useful, for example, for reverting the cellular phenotype(s)caused by the inserted insertional mutagen, which can be useful forconfirming that a particular change in cellular phenotype is caused by amutation induced by the insertion of the insertional mutagen. Thisapproach is also useful for removing certain sequences from the insertedinsertional mutagens, such as selectable markers, while leaving othersequences in the insertional mutagen, such as sequences that disrupt oneor more genes in the DNA in which the insertional mutagen has inserted.Any site-specific recombination system can be used that is capable ofdeleting or inverting an inserted insertional mutagen. Examples ofuseful recombination signals include loxP, FRT, and att which are usefulin conjunction with Cre, FLP (or FLPe) and PhiC31 recombinases,respectively (see, e.g., Hoess and Abremski, in: Nucleic Acids andMolecular Biology, vol. 4. Eds.: Eckstein and Lilley, BerlinSpringer pp.90(1990); Broach, et al., Cell 29:227(1982); Ishida et al., Nucl. AcidsRes. 27:e35 (1999); O"Gorman et al., Science 251:1351-1355 (1991);Bergemann et al., Nucl. Acids Res. 23:4451-4456 (1995); Sauer, B., Curr.Opin. Biotech. 5:521(1994); Schwenk et al., Nucleic Acids Res. 2002 Jun1; 30(11):2299-306; Schaft et al.; Genesis. 2001 Sep;31(1):6-10; Farleyet al.; Genesis, 2000 Nov-Dec;28(3-4):106-10; Rodriguez et al.; NatGenet. 2000 Jun;25(2):139-40; Buchholz et al.; Nat Biotechnol. 1998Jul;16(7):657-62; Olivares et al.; Gene. 2001 Oct 31;278(1-2:167-76; LeeL, Sadowski PD, J Biol Chem. 2001 Aug 17;276(33):31092-8; Kolb AF, AnalBiochem. 2001 Mar15;290(2):260-71; Araki et al., Nucleic Acids Res. 1997Feb 15;25(4):868-72; Albert et al., Plant J. 1995 Apr;7(4):649-59;Santoro et al., Proc Natl Acad Sci USA 2002 Apr 2;99(7):4185-90; Trinhet al., J Immunol Methods, 2000 Oct 20;244(1-2):185-93; Soukharev etal., Nucleic Acids Res. 1999 Sep 15;27(18):e21 and U.S. Patent Nos.4,959,317, 5,434,066, 5,888,732, 6,080,576 and 6,136,566; eachincorporated herein by reference for teaching vectors and methods ofsite specific recombination in mammalian cells). Other examples ofsuitable recombination sites for use in the insertional mutagens of thepresent invention include the attB, attP, attL, and attR sequences whichare recognized by the recombination protein βand by the auxiliaryproteins integration host factor (IHF), FIS and excisionase (Xis). SeeLandy, Curr. Opin. Biotech. 3:699(1993); see also U.S. Patent Nos.5,888,732, 6,143,557, 6,171,969 and 6,277,608, each of which isincorporated by reference herein in its entirety. Additional examples ofrecombination systems include Hin, Gin, Pin, Cin, and VDJ recombination,all of which are well-known in the art and which will be familiar to theordinarily skilled artisan. Other site-specific recombination systemsknown in the art would also be recognized as useful by the ordinarilyskilled artisan, and therefore can be used in accordance with themethods and compositions of the present invention. The site specificrecombination signals may be wildtype or mutant. Mutant signals can beused, for example, to control the reversibility of the recombinationreaction (reference Araki et al., Nucleic Acids Res. 1997 Feb15;25(4):868-72; Dale et al., Plant J. 1995 Apr;7(4):649-59; Trinh etal., J Immunol Methods, 2000 Oct 20;244(1-2):185-93; Soukharev et al.Nucleic Acids Res. 1999 Sep 15;27(18):e21 incorporated in its entirety).The site-specific recombinases used in accordance with this aspect ofthe invention may be wildtype, mutant, or fusion proteins. Examples ofmodified recombinases useful in this aspect of the invention include,but are not limited to, cell-permeable CRE and CRE-ER (Jo et al., NatureBiotech. 19:929-933 (2001); Vallier et al., Proc. Natl. Acad. Sci. USA98:2467-2472 (2001); each of which is incorporated by reference hereinin its entirety). The recombinases used in the invention can bedelivered to cells by infection or transfection of an expression vectorencoding the recombinase (Westerman et al., Proc. Natl. Acad. Sci. USA93:8971-8976 (1996), which is incorporated by reference herein in itsentirety), transfection of the protein (e.g., via electroporation; Ageret al., Radiat. Res. 128:150-156 (1991); Chung et al., Radiat. Res.125:107-113 (1991); each of which is incorporated by reference herein inits entirety), or through the use of a cell-permeable recombinase (Jo etal., Nature Biotech. 19:929-933 (2001); Vallier et al., Proc. Natl.Acad. Sci. USA 98:2467-2472 (2001); each of which is incorporated byreference herein in its entirety). Alternatively, the recombinase genemay itself be present on the insertional mutagen. The site-specificrecombinase gene and/or recombination site from any of these systems canbe included on the insertional mutagens of the invention, or can beintroduced into the host cell separately to achieve the desiredrecombination event.

[0150] Using insertional mutagens containing one or more recombinationsites, mutated cells or organisms (e.g., cells or organisms containingone or more mutated genes) produced by the methods of the invention canoptionally be analyzed to confirm that any change in phenotype observedin the mutated cell or organism is the result of at least oneinsertionally mutated gene. One such method involves the use ofsite-specific recombination to reverse the phenotypic change, typicallyby inducing a reversion of the mutation to the wildtype by deleting orinverting the inserted insertional mutagen. In one such embodiment,site-specific recombination signals recognized by specific recombinaseenzymes (e.g., the att/Int system from bacteriophage, the lox/Cre systemfrom bacteriophage P1, and the frt/FLP system from the Saccharomycescerevisiae 2μcircle plasmid) can be included on the insertional mutagen(see Figures 9A-9H for non-limiting examples of such insertionalmutagens containing site-specific recombination signals). Generally,such recombination sites are positioned on the insertional mutagen toallow the entire insertional mutagen, or a portion of the insertionalmutagen responsible for mutating the gene, to be removed from orinverted within the DNA into which the insertional mutagen has insertedby introducing the appropriate recombinase enzyme into the cell.Optionally, as discussed below, use of a negative selectable marker or areporter gene can facilitate identification or isolation of cells inwhich the mutagenic portion of the insertional mutagen has been deletedor inverted, and any change in cellular phenotype as a result of suchdeletion or inversion can be assessed so as to determine the phenotypiceffects of the insertional mutagenesis (e.g., reversal or alteration ofthe cellular phenotype that is obtained upon insertional mutagenesisindicates that at least one insertional mutation is likely responsiblefor the observed change in phenotype from the wildtype).

[0151] The invention can also be used to identify developmentallyregulated genes. In one embodiment, the presence of both a positiveselectable marker and a negative selectable marker in the sameinsertional mutagen can be used, for example, to select against activelyexpressed genes and for developmentally regulated genes. Alternatively,the presence of both a positive selectable marker and a negativeselectable marker can be used to select for actively expressed genesthat are down-regulated in response to developmental or environmentalcues. Vectors and methods for trapping and selecting for developmentallyregulated genes are well-known in the art (see, e.g., Gogos et al., J.Virol. 71:1644-1650 (1997); Wempe et al., Genome Biol. 2:research23.1-23.10 (2001); and Medico et al., Nature Biotech. 19:579-582 (2001);the disclosures of all of which are incorporated herein by reference intheir entireties for these vectors and methods). Other methods foridentifying trapped developmentally regulated genes in accordance withthe invention involve the use of insertional mutagens that function asgene trap vectors such as those depicted in Figures 9B, 9C and 9H. Thesewill comprise, for example, at least two site-specific recombinationsignals (e.g., at least two lox sites (e.g., loxP), at least two attsites (e.g., attP, attB, attL and/or attR), at least two FRT sites, orthe like), which flank the positive selectable marker (as in Figure 9B),the negative selectable marker (as in Figure 9C), or both the positiveand negative selectable markers (as in Figures 9G and 9H).

[0152] In a first such embodiment, the insertional mutagens depicted inFigures 9B, 9H, 11, and 13, can be integrated using standard methods ofintroduction of nucleic acid molecules into host cells that arewell-known in the art and that therefore will be familiar to theordinarily skilled artisan. Once the insertional mutagen has beenintegrated, cells can be selected based on the positive selectablemarker carried by the insertional mutagen (and therefore integrated intoDNA in the host cell). Surviving clones will contain the insertionalmutagen integrated into a transcriptionally active gene, since it isonly in such cells that the positive selectable marker will also beexpressed. Cells can then be treated to delete the positive selectablemarker (Fig. 11) or invert the positive marker (Fig. 13 legend) andcreate an operable linkage between the trapped gene and the negativeselectable marker. In certain such aspects of the invention, this resultis obtained through a site-specific recombination reaction: cells aretreated with a site-specific recombinase, the identity of which willdepend upon the specific recombination site used in construction of theinsertional mutagen (e.g., Cre recombinase is used with loxrecombination sites; Int recombinase is used with att recombinationsites; FLP recombinase is used with frt recombination sites; etc.). Suchtreatment results in recombination between opposing recombination sites(see, e.g., Figure 9B), thereby deleting or inverting the positiveselectable marker from (and operably linking the negative selectablemarker and the trapped gene) the genome of the cell. Cells can then becultured under new conditions and/or treated with selection agents, andcells can be selected for lack of expression of the negative selectablemarker to identify cells in which transcription of the trapped gene hasbeen reduced or eliminated (e.g., cells that survive the negativeselection). This process is illustrated schematically in Figureanddescribed in Fig. 13 legend..

[0153] In another such embodiment, the insertional mutagens depicted inFigures 9C and 9H can be integrated using standard methods ofintroduction of nucleic acid molecules into host cells that arewell-known in the art and that therefore will be familiar to theordinarily skilled artisan. Once the insertional mutagens have beenintegrated, cells can be selected based on the negative selectablemarker carried by the insertional mutagen (and therefore integrated).Cells in which the insertional mutagen has integrated into (e.g., thathave trapped) a transcriptionally active gene will die during selectionsince it is only in such cells that the negative selectable marker willalso be expressed, whereas cells in which the insertional mutagen hasintegrated into a transcriptionally silent region of the genome willsurvive. Cells can then be treated to delete the negative selectablemarker and create an operable linkage between the trapped gene and thepositive selectable marker, for example using site-specificrecombination as outlined above. Such treatment results in recombinationbetween opposing recombination sites (see, e.g., Figure 9C), therebydeleting (Fig. 12) or inverting (Fig. 13) the negative selectable markersuch that the positive selectable marker becomes operably linked to thetrapped gene. Cells can then be cultured under new conditions or treatedwith agents capable of inducing gene expression, and cells can beselected for expression of the positive selectable marker to identifycells in which transcription of the trapped gene is increased (e.g.,cells that survive the positive selection). This process is illustratedschematically in Figures 12 and 13.

[0154] In certain other embodiments of the invention, the insertionalmutagens can additionally or alternatively comprise one or more reportergenes, also known as screenable markers. A reporter gene is a gene thatencodes an expression product that can be detected in the cell. Inaccordance with the invention, the reporter gene may be expressed from apromoter on the insertional mutagen, or from a promoter in the DNA intowhich the insertional mutagen is inserted. Detection of the reportergene allows the artisan to screen for cells that are or are notexpressing the reporter gene. In the present invention, reporter genesallow insertions to be detected. In addition, reporter genes can bepositioned on the insertional mutagen to allow screening for insertionevents that occur in transcriptionally active or silent regions of thegenome (see Figures 2-6). Reporter genes suitable for use in accordancewith the invention can be any gene that encodes an expression productfor which an assay exists. Examples of such suitable reporter genesinclude, but are not limited to, enzymes, structural proteins, cellsurface proteins, and fluorescent proteins. Specific reporter genesknown in the art include β-lactamase, β-galactosidase, luciferase,chloramphenicol acetyl transferase, green fluorescent protein and itsderivatives, yellow fluorescent protein and its derivatives, bluefluorescent protein and its derivatives, cyan fluorescent protein andits derivatives, and red fluorescent protein and its derivatives. Manyother reporter genes are known in the art and would be recognized by theartisan as being useful in the present invention.

[0155] Assays for detecting reporter genes include, but are not limitedto, enzyme activity assays, cell microfluorimetry orfluorescence-activated cell sorting (FACS®), magnetic bead cell sorting,ELISA, ELISA Spot, transcriptional reporter assays, and cellularphenotypic assays such as proliferation, transformation, morphology, andthe like.

[0156] Reporter genes can be used optionally in place of or in additionto the site specific recombination sequences to identify developmentallyregulated genes and to revert phenotypes.

[0157] In certain other embodiments of the invention, the insertionalmutagens can additionally or alternatively comprise one or moreselectable markers and one or more reporter genes. The one or moreselectable markers and one or more reporter genes can be present as afusion gene or as two discrete open reading frames. Any combination ofselectable markers and reporter genes can be used, including thosedetailed above. An example of a useful selectable reporter gene fusionis β-geo, a fusion of the neomycin resistance gene and theβ-galactosidase gene. Other fusion genes known in the art, and whichtherefore are familiar to the ordinarily skilled artisan, can also beused in the present invention.

[0158] In certain other embodiments of the invention, the insertionalmutagens can additionally or alternatively comprise one or more spliceacceptor sequences. Upon introduction into the target DNA, the one ormore splice acceptor sequences can become paired with one or more splicedonor sequences in the target DNA, thereby directing splicing from thegene in the DNA to the inserted insertional mutagen. This splicing, inturn, facilitates mutation of a gene in the target DNA through thecreation of a fusion mRNA molecule. Any sequence capable of functioningas a splice acceptor site can be used in accordance with this aspect ofthe present invention. The splice acceptor site can be naturallyoccurring or non-naturally occurring. Splice acceptor sites and methodsfor testing the splicing activity of candidate or putative spliceacceptors are known in the art, and therefore will be familiar to theordinarily skilled artisan. In human cells, splice acceptor sites have acharacteristic sequence represented as: YYYYYYYYYYNYAG, wherein Ydenotes any pyrimidine and N denotes any nucleotide (see Nucleic AcidsResearch 19:3715-3799 (1991)).

[0159] In other embodiments of the invention, the insertional mutagenscan additionally or alternatively comprise one or more splice donorsequences. Upon introduction into the target DNA, the one or more splicedonor sequences can become paired with one or more splice acceptorsequences in the target DNA, thereby directing splicing from thepolynucleotide to a gene in the target DNA. This splicing, in turnfacilitates mutation of the gene through the creation of a fusion mRNAmolecule. The splice donor site may optionally be paired with a spliceacceptor site on the insertional mutagen. Typically, in such aconfiguration, the order of these elements will be the splice acceptorfollowed by the splice donor (see Figures 1H-1J, 2D and 2H fornon-limiting examples). The splice donor site may optionally be operablylinked to a promoter on the insertional mutagen to produce apromoter-splice donor 3' gene trap (see Figures 5B-5E for non-limitingexamples of such vectors). Any sequence capable of functioning as asplice donor site can be used. The splice donor site can be naturallyoccurring or non-naturally occurring. Splice donor sites and methods fortesting splicing activity of candidate or putative splice donor sitesare known in the art, and therefore will be familiar to the ordinarilyskilled artisan. In human cells, splice acceptor sites have acharacteristic sequence represented as: (A/C)AG GURAGU, wherein Rdenotes a purine nucleotide (see Nucleic Acids Research 19:3715-3798(1991)). The insertional mutagen may contain one or more exon sequences.These can be naturally occurring or nonmade, as by recombinant DNA orsynthetic methods. The exons may be derived from eukaryotic genes.Further, the one or more exons can be in tandem.

[0160] In other embodiments, the insertional mutagens can additionallyor alternatively comprise one or more sequences that direct the additionof 5' or 3' polynucleotide tails on mRNA molecules transcribed from theDNA into which the insertional mutagen integrates. Such sequence canencode any polynucleotide tail, such as poly (A) tails, poly (G) tails,poly (U) tails, poly (C) tails, poly (I) tails, and the like. In oneembodiment, the insertional mutagen comprises one or morepolyadenylation signals that direct the addition of poly (A) tails onmRNA molecules transcribed from the DNA into which the insertionalmutagen integrates. Polyadenylation signals can be derived fromnaturally occurring or non-naturally occurring sequences. Examples ofuseful polyadenylation signals include, but are not limited to, thosederived from SV40 genes, growth hormone genes (e.g., bovine growthhormone), β-globin genes, actin genes, serum albumin genes, andretrovirus genes. Other polyadenylation signals are known in the art andwill therefore be familiar to the ordinarily skilled artisan.

[0161] In certain other embodiments of the invention, the insertionalmutagens can additionally or alternatively comprise one or more internalribosomal entry sites (IRES). The IRES allows translation of internalopen reading frames and are useful for expressing open reading frameslocated on the insertional mutagens upon integration intotranscriptionally active genes. In certain embodiments, the ORF on theinsertional mutagen is a selectable marker and/or reporter gene. AnyIRES can be used to express an ORF located on the insertional mutagen.Examples of useful IRESs and methods of measuring IRES activity areknown in the art (see, for example, Zhou et al., PNAS 98:1531-1536(2001), Owens et al., PNAS 99:1471-1476 (2001); Venkatesan et al., MolCell Biol. 8: 2826-2837 (2001); Jackson et al., Trends Biochem. Sci. 15:477-483 (1990); and Jang et al., J. Virol. 62:2636-2643 (1988); eachincorporated herein by reference for teaching IRES sequences and methodsfor measuring IRES activity).

[0162] In certain other embodiments of the invention, the insertionalmutagens can additionally or alternatively comprise one or moretransposon signals. 1. Cui et al., J Mol Biol. 2002 May17;318(5):1221-35; 2. Izsvak et al., J Biol Chem. 2002 Jun 24; 3. Dupuyet al., Proc Natl Acad Sci U S A. 2002 Apr 2;99(7):4495-9; 4. Horie etal., Proc Natl Acad Sci U S A. 2001 Jul 31;98(16):9191-6; 5. Dupuy etal.,Genesis. 2001 Jun;30(2):82-8; 6. Fischer et al., Proc Natl Acad SciU S A. 2001 Jun 5;98(12):6759-64; 7. Ivics et al.,Cell. 1997 Nov14;91(4):501-10. Other transposons also function in mammals: 8.Zagoraiou et al., Proc Natl Acad Sci U S A. 2001 Sep 25;98(20):11474-8;9. Sherman et al., Nat Biotechnol. 1998 Nov;16(11):1050-3; 10. Kawakamiet al., Proc Natl Acad Sci U S A. 2000 Oct 10;97(21):11403-8; 11. Fadoolet al., Proc Natl Acad Sci U S A. 1998 Apr 28;95(9):5182-6; 12. PlasterkRH, Cell. 1993 Sep 10;74(5):781-6; 13: Kaufman et al.,Nucleic Acids Res.1991 Nov 25;19(22):6336; 14. Rubin et al., Nucleic Acids Res. 1983 Sep24;11(18):6341-51; 15. Spradling et al., Science. 1982 Oct22;218(4570):341-7; Ac and Ds and other plant transposons transpose,integrate and are used as insertional mutagens in plants: 16. Greveldinget al, Proc Natl Acad Sci U S A. 1992 Jul 1;89(13):6085-9; 17. WalbotV., Curr Opin Plant Biol. 2000 Apr;3(2):103-7; 18. Pereira et al,Methods Mol Biol. 1998;82:329-38; 19. Cooley et al., Mol Gen Genet. 1996Aug 27;252(1-2):184-94; 20. Bhatt et al, Plant J. 1996 Jun;9(6):935-45.P element transposon can integrate broadly throughout genomes: 21.Kassis et al., Proc Natl Acad Sci U S A. 1992 Mar 1;89(5):1919-23; 22.Berg et al., Genetics. 1991 Mar;127(3):515-24; 23. Tower et al.,Genetics. 1993 Feb;133(2):347-59; 24. Cooley et al., Prog Nucleic AcidRes Mol Biol. 1989;36:99-109; 25. Cooley et al., Science. 1988 Mar4;239(4844):1121-8; 26. Spradling et al., Proc Natl Acad Sci U S A. 1995Nov 21;92(24):10824-30.

[0163] Transposon signals allow the insertional mutagens to insert intothe DNA by expressing or otherwise introducing transposase in the cellwith the insertional mutagen. In a preferred embodiment, the insertionalmutagen is first introduced into DNA in a cell, and subsequentlytransposed, or "hopped," in order to insertionally mutate one or moregenes. This can be done in vitro, in vivo, and in situ. Methods fortransposing vectors in situ are well known in the art (see, for example,Lucklow et, J.67:4566(1993); Ivics et al., Cell 91:501-510 (1997); andLuo et al., PNAS 95:10769-10773 (1998); each incorporated herein byreference for teaching vectors and methods of transposition thereof).

[0164] There are many transposon systems and transposon signals known inthe art that are useful in the present invention. These include TY fromyeast (e.g., TY1; see Devine and Boeke Nucl. Acids Res. 22:3765(1994),which is incorporated by reference herein in its entirety), P-elements,Hobo, Minos, and Manner from Drosophila, Tn5, Tn7, T0, Tn10, and Mu frombacteria, En/Spm from maize, and TCI/Mariner (and its derivatives, suchas Sleeping Beauty) and Minos from mouse and C. elegans. Many othertransposon systems are known in the art and would be recognized by theartisan as useful in the present invention. In addition, modifiedtransposon signals and mutant transposases with enhanced efficiency havebeen described and would be useful in the presentAny of the insertionalmutagens described herein for insertion can be produced as viralvectors, such as retroviral vectors (including lentivirus),Herpesviruses vectors (such as Epstein-Barr virus, cytomegalovirus(CMV), Herpes zoster, and Herpes simplex), papillomavirus, picornavirus,papovavirus (such as polyoma vectors and SV40), adenovirus,adeno-associated virus, and hepatitis virus. Particularly preferredvectors are retroviral. Viruses have the advantage of efficientlyintroducing the insertional mutagens into a cell, and, in the case ofsome viruses, facilitating efficient delivery of the insertional mutagento the cell, and integration of the insertional mutagen into DNA in acell.

[0165] Retroviral vectors of the invention and of use in the methods ofthe invention can contain retroviral LTRs, packaging signals, and anyother sequences that facilitate creation of infectious retroviralvectors. Retroviral LTRs and packaging signals allow the insertionalmutagens of the invention to be packaged into infectious particles anddelivered to the cell by viral infection. Methods for making recombinantretroviral vectors are well known in the art (see for example, Brenneret al., PNAS 86:5517-5512 (1989); Xiong et al., Developmental Dynamics212:181-197 (1998) and references therein; each incorporated herein byreference). In preferred embodiments, the retroviral vectors used in theinvention are reverse orientation vectors, meaning that the genemutation element in the insertional mutagen is in the opposite directionof viral transcription. The retroviral vectors can also be SelfInactivating viruses (SIN viruses). SIN viruses are nottranscriptionally active upon insertion. Methods for making SIN virusesare known in the art (see for example, Hawley et al., PNAS 84:2406-2410(1987); Brenner et al., PNAS 86:5517-5512 (1989); and Lih et al., Gene164:289-294 (1995); each incorporated herein by reference for teachingvectors). Retroviral LTRs and packaging signals can be selectedaccording to the intended host cell to be infected. Examples ofretroviral sequences useful in the present invention include thosederived from Murine Moloney Leukemia Virus (MMLV), Avian Leukemia Virus(ALV), Avian Sarcoma Leukosis Virus (ASLV), Feline Leukemia Virus (FLV),and Human Immunodeficiency Virus (HIV). Other viruses known in the artare also useful in the present invention and therefore will be familiarto the ordinarily skilled artisan.

[0166] In certain other embodiments, the insertional mutagens canadditionally or alternatively comprise one or more bacterial sequencesuseful for propagating the insertional mutagens in prokaryotic oreukaryotic cells. Thus, the insertional mutagens of the invention cancontain, for example, one or more antibiotic resistance markers, and/orone or more other art known sequences useful for propagating andanalyzing the insertional mutagens.

[0167] Any of the insertional mutagens described herein can further oralternatively comprise one or more 3' gene trap. A number of 3' genetraps have been described and are useful in the present invention (seee.g., Harrington et al., WO 99/15650; Zambrowicz et al., U.S. Patent No.6,080,576; Sands et al., U.S. Patent No. 6,136,566; Niwa et al., J.Biochem. 113:343-349 (1993); Yoshida et al., Transgenic Research4:277-287 (1995); all incorporated herein by reference in its entiretyfor teaching 3′ gene traps). The 3' gene trap can be used to recoverexons in the target DNA that are downstream of the insertional mutageninsertion site. Optionally, it can also be used to activate RNA orprotein expression from genes in the target DNA. When a 3' gene trapcassette is contained in a insertional mutagen of the present invention,it can be located upstream or downstream of a mutagenic portion of theinsertional mutagen. In preferred embodiments, the 3' gene trap islocated 3' of the mutagenic portion of the insertional mutagen.

[0168] Further, any of the insertional mutagens described herein canfurther or alternatively comprise one or more 5' gene trap cassettes. Anumber of 5' gene traps have been described and are useful in thepresent invention (see e.g., Zambrowicz et al., U.S. Patent No.6,080,576; Sands et al., U.S. Patent No. 6,136,566; Niwa et al., J.Biochem. 113:343-349 (1993); Yoshida et al., Transgenic Research4:277-287 (1995); all incorporated herein by reference in its entiretyfor teaching 3′ gene traps). The 5' gene trap can be used to recoverexons in the target DNA that are downstream of the insertional mutageninsertion site. Optionally, it can also be used to activate RNA orprotein expression from genes in the target DNA. When a 5' gene trapcassette is contained in a insertional mutagen of the present invention,it can be located upstream or downstream of a mutagenic portion of theinsertional mutagen. In preferred embodiments, the 5' gene trap islocated 5' of the mutagenic portion of the insertional mutagen.

[0169]Integration of the Insertional MutagensThe insertional mutagens ofthe invention can be introduced into a cell and integrated into DNA byany method known in the art. In a preferred embodiment, they areintroduced by transfection. Methods of transfection include, but are notlimited to, electroporation, particle bombardment, calcium phosphateprecipitation, lipid-mediated transfection (e.g., using cationiclipids), micro-injection, DEAE-mediated transfection, polybrene mediatedtransfection, naked DNA uptake, and receptor mediated endocytosis.

[0170] In another preferred embodiment, the insertional mutagens areintroduced by viral transduction or infection. Suitable viral vectorsuseful in the present invention include, but are not limited to,adeno-associated virus, adenovirus vectors, alpha-herpesvirus vectors,pseudorabies virus vectors, herpes simplex virus vectors and retroviralvectors (including lentiviral vectors). Methods for making and usingviral vectors are described above and elsewhere herein, and arewell-known in the art and therefore familiar to the ordinarily skilledartisan (see, for example, Viral Vectors: Gene Therapy and NeuroscienceApplications E. Caplitt and Loewy, Academic Press, San Diego (1995);incorporated herein by reference for teaching viral vectors and methodsof using such vectors for introducing and expressing polynucleotides ofinterest).

[0171] In a preferred such embodiment, the vectors are retroviralvectors (including lentiviral vectors) and are introduced into the cellby infection. Vectors containing viral LTRs and packaging signals aredescribed above. Methods for packaging retroviral vectors are also knownin the art and can be used in the present invention (see, for example,U.S. Patent No. 5,449,614, the disclosure of which is incorporatedherein by reference in its entirety for teaching vectors, packaging celllines, and methods of making and packaging viral vectors).

[0172] Following induction of an endogenous insertional mutagen or itsintroduction into a cell by transfection or infection, the insertionalmutagens of the invention integrate into the genome of the cell. Theinsertional mutagen can integrate into the target DNA by any methodincluding, but not limited to, non-homologous recombination includingretroviral insertion and transposition, site-specific recombination,homologous recombination and the like. In certain preferred embodiments,the insertional mutagen integrates by nonrecombination (e.g.,integration by DNA end-joining, retroviral insertion, or transposition).

[0173] In certain preferred embodiments of the invention, the cell canbe treated with one or more DNA-breaking agents prior to, during, orfollowing induction or introduction of the insertional mutagen into thecell. DNA-breaking agents increase the efficiency of integration.Examples of DNA-breaking agents suitable for use in accordance with thisaspect of the invention include, but are not limited to, γ-radiation,X-ray irradiation, UV irradiation, bleomycin, peroxides, and restrictionenzymes. Other agents known to break DNA in living cells can also beused. Methods of using DNA breaking agents to enhance insertionalmutagen insertion have been described (see, e.g., Harrington et al., WO99/15650, incorporated herein by reference for teaching methods ofenhancing nonhomologous recombination).

[0174] In one embodiment, the initial integration is not the mutagenicevent. Where the insertional mutagen contains a transposition signal,after the initial integration, the methods of the invention can becarried out by inducing an endogenous insertional mutagen to transpose(hop) to a new location, where the mutagenic event(s) can occur. Methodsfor transposing vectors in situ are well known in the art and thereforewill be familiar to the ordinarily skilled artisan (see, for example,Ivics et al., Cell 91:501-510 (1997); and Luo et al., PNAS95:10769-10773 (1998); each incorporated herein by reference forteaching transposition vectors and methods).

[0175] A variety of genes can be mutated using the methods of theinvention. For example, known genes, including disease-causing genes(e.g., oncogenes, integrated viral genes (including HIV), genes causinggenetic abnormalities such as cancers, multiple sclerosis, Alzheimer"sdisease, diabetes, muscular dystrophy, ALS, Gaucher"s Disease, Tay-Sachsdisease, hemophilia, β-thalassemia, cystic fibrosis, sickle cell trait,and the like) and normal genes (imparting any phenotype to a cell ororganism) can also be mutated using the methods of the invention. Inanother embodiment, genes which have been previously unknown orincompletely characterized can be mutated using the methods of theinvention. In another embodiment, genes can be mutated that are known orcharacterized but which were not known to be correlated to a desiredphenotype produced by the mutagenesis methods of the invention. Theinvention thus provides a way to identify, isolate and characterizepreviously unknown or incompletely characterized genes in a variety ofeukaryotic cells, and to examine the phenotypic importance of such genesby examining the effects on cellular phenotype when the genes aremutated.

[0176] It is also possible to have multiple (more than one per cell)insertional mutagens in each target cell to increase the probabilitythat at least one gene will be mutated in the cell. Thus, cells createdby the present methods can contain one or more integrated insertionalmutagen. In certain embodiments, each of the target cells will contain1-10 insertional mutagens, or 10 or more insertional mutagens. Two ormore insertional mutagens in a single cell has the advantage of reducingthe total number of cells that must be screened to identify a cell witha mutation of a desired gene or of a gene(s) that causes a desiredphenotype.

[0177] The number of insertional mutations that would be useful dependson the size of the genome of the host cell, its ploidy, gene structure,the average insertion window associated with gene mutation (e.g., sizeof the gene), the amount of genome coverage that is desired (e.g., thepercentage of genes that are to be insertionally mutated), thepropensity for integration of the insertional mutagen into genes, andthe number of genes capable of producing a desired phenotype whenmutated. In higher eukaryotic organisms, the genome is typically large.In mouse and human cells, for example, the haploid genome is estimatedto be 3 x 10⁹ base pairs (6 billion basepairs for the diploid genome).Therefore, 10⁶ insertions will result in an insertional mutagenic eventfrequency of 1 insertion per 3,000 base pairs. Assuming that an averagehuman gene is approximately 25,000 base pairs, and assuming randominsertion, 10⁶ insertions at a frequency of 1 in 3,000 base pairs will,on average, result in the mutation of one copy of each gene at leastonce. In practice, it can be necessary to create a larger or smallernumber of insertions if integration is found not to be random. Forexample, it can be necessary to produce 10⁷ or more random insertions toinsertionally mutate the majority of genes at least once. It is alsouseful in certain situations or for certain applications to create fewerinsertions. In preferred embodiments, an insertion library containing atleast 10,000 insertions is created. In highly preferred embodiments, aninsertion library containing at least 100,000 insertions is created. Inthe situation where the desire is to focus mutagenesis on those genesthat can be transcriptionally active in the cell under study, then useof insertional mutagens that show a bias for insertion into active genes(e.g., retroviruses or transposons) can enable the creation of librariesof 10⁴-10⁵ members that contain knockouts of all active genes.Sincegenome sizes vary, it is possible to rely on general guidelines fordetermining the number of insertions necessary to create a library of agiven complexity. As a general guideline it is useful to create 1insertion per 1000 to 10,000 base pairs of the host cell genome. In oneembodiment, 1 insertion is created per 30,000 base pairs of host cellgenome. In highly preferred embodiments, the insertion frequency isadjusted to create at least one mutation per gene in the library. Itshould be understood that these are general guidelines and that otherinsertion frequencies are possible and recognized by those skilled inthe art.

[0178] In embodiments where the insertional mutagen contains aselectable marker or reporter gene, cells can be selected forintegration by selecting for expression of the selectable marker. If theselectable marker is expressed from a promoter on the insertionalmutagen (see, for example, Figure 14), then any cell containing anintegrated insertional mutagen should be recovered. If the selectablemarker is not expressed from a promoter on the insertional mutagen, butis expressed from an upstream promoter on the target DNA (see Figures2-6 for examples), then any cell containing the insertional mutagenintegrated into a transcriptionally active gene can be selected for oragainst depending on whether a positive or negative selectable marker,respectively, is being used. Integration into transcriptionally activegenes is desirable because in instances where the cells are going to bescreened for a phenotype, mutation of a transcriptionally active gene ismore likely to give a phenotype than mutation of a transcriptionallyinactive gene. Alternatively, selection against a transcriptionallyactive gene can be useful for removing cells that have insertionallymutated a transcriptionally active gene so that other genes can bestudied. For example, after removing cells containing mutatedtranscriptionally active genes, cells can be treated with agents thatcause a change in gene expression within the cell and the artisan cannow screen for phenotypes that result from mutation of genes that werepreviously silent but became active. Reporter genes can also optionallybe used to screen for integration into transcriptionally active orsilent genes.

[0179]Library ConstructionThe two mutagenesis methods can be carried outon one cell. The cell can then be screened for mutation of a specificdesired gene or for a desired phenotype that results from mutation ofone or more genes.

[0180] In one embodiment, insertional mutagenesis is carried out on asingle physicochemically mutated clone of cells. For example, a singlephysicochemically mutated cell can be expanded and used for insertionalmutagenesis. This creates a library of cells that are mutagenized byboth methods. Typically, for mammalian cells, between 1 and 10⁷ or moregene trap insertions are created for each physicochemically mutatedclone. A gene trap insertion is an instance where genomic integration ofthe insertional mutagen has become operably linked to a constitutivelyor conditionally active endogenous gene such that exonic sequences inthe insertional mutagen are incorporated into the transcripts of theendogenous gene. Many of these gene trap insertions will disrupt thenormal function of the insertionally mutagenized genes. In someembodiments this range would be to 10² to 10⁴ insertions per mutatedclone. In certain preferred embodiments, the number of insertion mutantscreated for each physicochemically mutated clone ranges between 10⁴ and10⁶, and often between 10⁴ and⁵. In one embodiment, after theinsertional mutagenesis, the resulting library can be screened forintegration of one or more of the insertional mutagens. In anotherembodiment, the resulting library is screened for mutation of a specificgene of interest or for a phenotype that results from mutation of a geneof interest, or for a phenotype that results from mutation of a genethat has not been previously identified or not previously known to becorrelated with the phenotype. The mutant cells produced by thesemethods of the invention also can be cloned. The mutant cells thatexhibit the phenotype derived from the combined mutagenesis can becloned, for example to generate a purified population for furthermanipulation or use.

[0181] In another embodiment, insertional mutagenesis is carried out onmore than one physicochemically mutated clone of cells. For example,more than one physicochemically mutated cell can be expanded forinsertional mutagenesis. The number of physicochemically mutated clonescan range from 1 to 10⁶ or more. In preferred embodiments, the number ofphysicochemically mutated clones used to create the insertion mutants isbetween 1 and 100,000 and more preferably between 1 and 10,000. Inhighly preferred embodiments, the number of physicochemically mutatedclones used to create the insertion mutants is between 1 and 1000.Useful ranges include approximately 2, 5, 10, 25, 50, 100, 200, 400,600, and 800 clones. Guidelines for assessing physicochemical mutationfrequency and selecting appropriate numbers of mutated cells arediscussed above in the section describing physicochemical mutagenesis.The selected number of physicochemically mutated cells can be expandedand used to create a library of cells that are mutagenized by the twomethods. Typically, for mammalian cells, between 1 and 10⁸ or moreinsertions are created for each physicochemically mutated clone present.In preferred embodiments, the number of insertion mutants created foreach physicochemically mutated clone ranges between 10⁴ and 10⁶, andoften between 10⁴ and 10⁵. For example, without limitation, if 100physicochemically mutated clones are used to create a library, then alibrary with 10⁴ insertions per clone would result in the creation of alibrary with 10⁶ clones (100 physicochemically mutated clones x 10⁴insertionally mutated clones). In one embodiment, the resulting librarycan be screened for integration of one or more insertional mutagensafter the insertional mutagenesis. In another embodiment the resultinglibrary can be screened for mutation of a gene of interest, for aphenotype that results from mutation of a gene of interest, or for aphenotype that results from a mutation of a gene not previouslyidentified or not known to be correlated with the phenotype. The mutantcells produced by these methods of the invention also can be cloned.

[0182] It is also possible to make the library by insertionalmutagenesis and then use that library for physicochemical mutagenesis.In one embodiment, one or more cells is mutated by integration of one ormore insertional mutagens. Typically, in mammalian cells, theinsertional mutagen is inserted into 1 to 10⁷ or more cells. Inpreferred embodiments, the insertional mutagen is inserted into between10⁴ to 10⁶ cells, and often into between 10⁴ to 10⁵ cells. Cellscontaining the integrated insertional mutagens are thenphysicochemically mutated. With either procedure, in one embodiment,clones are expanded prior to physicochemical mutagenesis. Preferably,between 50 and 10,000 genes are mutated in each physicochemicallymutated cell. In certain embodiments, approximately 1,000 genes aremutated in each target cell. Once created, thephysicochemically/insertionally mutated library of cells can be screenedfor mutation of a gene of interest or for a phenotype that results frommutation of a gene of interest, or that results from mutation of a genethat has not been previously identified or known to be correlated to thephenotype. The mutant cells produced by these methods of the inventionalso can be cloned.

[0183]Library ScreeningLibraries of mutant cells can be screened formutation of a desired gene. Gene expression levels or gene productactivity could be assayed or another phenotype that is associatedspecifically with the desired gene could be assayed. The assays can beused to identify cells with reduced or missing gene expression orfunction or with increased or restored gene expression or function. Thetag is still useful in this embodiment. It can be used to verify thatthe mutation is in the desired gene or to ascertain if the desired geneis improperly expressed because of a mutation in a separate gene. Thetag could also be useful as a way to isolate the mutated cell or cloneof cells from a large number of cells when there is no assay that issufficiently sensitive.

[0184] Examples of useful assays to detect a desired gene include, butare not limited to, ELISA, ELISA spot assays, PCR (e.g., rtPCR),transcription reporter assays, western blot, northern blots, Southernblots, electrophoretic mobility shift assays, transcriptional profiling(e.g., using gene chips), enzyme assays (e.g., protease, kinase,phosphatase, hydrolase, and other known assays), ligand binding assays,and Fluorescence Activated Cell Sorting (FACS®) and magnetic bead cellsorting.

[0185] Libraries of mutant cells produced by the present invention canalso be screened for desired phenotypes. Cells displaying the desiredphenotype can then be used to identify one or more mutant genesresponsible for the phenotype where the mutagenic polynucleotidecomprises a tag that tags the mutated gene. This approach can be used,for example, to identify mutant genes that play a role in a cellular orbiochemical process. By such methods of the invention, changes in avariety of cellular phenotypes that may be associated with geneticmutations may be analyzed, including without limitation: cellproliferation, cell transformation, cell migration, celldifferentiation, signal transduction, cell morphology, cell transport,protein degradation, apoptosis, chemoresistance, chemosensitivity,inflammatory response, nuclear translocation of proteins, proteinsecretion, cellular activation, gene activation, protein expression,receptor activation, and metastasis. See also the previous list above.Many other assays are known in the art that the ordinarily skilledartisan would recognize as useful in the present invention.

[0186] The methods of the present invention can also be used in screensfor the presence of conditional mutations in cells or organisms.Conditional mutations allow a mutation in a given gene to remain silentuntil a phenotypic screen (often dependent upon expression of the gene)is performed. This approach is particularly advantageous in situationswhere, for example, a mutation is toxic to the host cell, creates aslow-growth or no-growth phenotype, kills the cell, induces terminaldifferentiation, or is otherwise deleterious to the cell. Examples ofsuch conditional mutations that may be used in accordance with, or thatmay be detected by, the methods of the present invention include but arenot limited to temperature sensitive mutations (heat- or cold-sensitivemutations), sensitivity to chemicals such as dimethylsulfoxide,site-specific recombination in vitro or in vivo, translationread-through, and the like.

[0187]Uses of Mutated CellsOnce a cell with a desired phenotype isidentified, the mutated gene can be identified via the tag present onits protein or mRNA or by analyzing the genomic integration site of theinsertional mutagen, as discussed below. Methods for isolating thetagged gene include, but are not limited to, 5' RACE, inverse PCR, andcDNA library construction and hybridization. Methods for cloning genesthat have been mutated or activated are known in the art (see forexample, Harrington et al., U.S. Patent Application No. 09/276,820 filedMarch 26, 1999; Zambrowicz et al., U.S, Patent No. 6,080,576; Sands etal., U.S. Patent No. 6,136,566; Niwa et al., J. Biochem. 113:343-349(1993); Yoshida et al., Transgenic Research 4:277-287 (1995); Baker etal., Dev. Biol. 185:201-214 (1997); each incorporated herein byreference for teaching methods of identifying genes mutated by themutagenic polynucleotides).

[0188] The present invention can also be used to discover novel drugsand drug targets for use in diagnosing, treating or preventing a varietyof diseases and physical disorders. For example, cDNA molecules andgenomic fragments containing mutated genes of interest can be used toproduce a gene product in vitro or in a cell or animal, to screen drugs,develop new diagnostic methods or assays related to the genotype orphenotype of interest, or to express proteins for therapeutic use (e.g.,the gene may encode a protein such as erythropoietin, that can beadministered to patients to treat a condition). The mutant gene or geneproduct also can be used to identify the corresponding wild-type gene orgene product. The wild-type gene can be used to produce a wild-type geneproduct in vitro or in a cell or animal, to screen drugs, to develop newdiagnostic methods or assays related to the genotype or phenotype ofinterest, or to express proteins for therapeutic use (e.g., the gene mayencode a protein such as erythropoietin that can be administered topatients to treat a condition).

[0189] Mutated cells made using the present invention can be used indrug screening. For example, mutated cells displaying a therapeuticallyrelevant genotype or phenotype can be isolated from a library of mutatedcells. Once isolated, the mutated cells can be exposed to test compoundsto identify compounds that inhibit, further stimulate, or otherwisemodulate the genotype or phenotype of interest. By carrying out thisprocess, drugs and/or drug leads can be identified. Examples ofphenotypes relevant to drug screening include, but are not limited to,apoptosis, cell proliferation, chemosensitivity, chemotherapeuticresistance, cell migration, cell activation (e.g., T cell activation),cell transformation, metastasis, cellular differentiation, signaltransduction, transcriptional activation, protein expression, proteindegradation, protein secretion, and other phenotypes known in the artthat will be readily apparent to the ordinarily skilled artisan.

[0190] Mutated cells prepared according to the present invention canalso be used for manufacturing or other commercial purposes. Forexample, cells of the invention displaying high growth rates, highprotein expression levels, high levels of protein secretion, optimizedpost-translational modification of expressed proteins, ability to growin serum-free or other defined or inexpensive culture media, etc., offeran advantage in commercial applications such as in manufacturingproteins, foods, beverages, therapeutics, etc.

[0191] Mutated cells prepared according to the methods of the presentinvention can also be used to study gene function in vivo. In oneembodiment, cells mutated by the present invention are introduced intoan animal by adoptive transfer. Cells displaying a desired phenotype inthe animal can then be recovered and isolated. Alternatively, mutatedcells that display a desired phenotype in culture can be introduced intoan animal by adoptive transfer to study the in vivo phenotype of thecell. Examples of in vivo assays include, but are not limited to, tumorformation, metastasis, graft versus host disease, autoimmune disease,transplant rejection, reconstitution of missing or non-functional celltypes (e.g., bone marrow transplantation), cell differentiation, andother assays known in the art. Methods for introducing cells into ananimal by adoptive transfer are well known in the art (see, for example,Roth et al., J Exp Biol. 200:2057-2062 (1997); Mosier Adv Immunol.50:303-325 (1991); Mule et al., J. Immunother. 12:196-198 (1992); eachincorporated herein by reference for teaching methods and uses ofadoptive transfer). Optionally, the mutated gene can be identified fromthe mutated cell.

[0192] In another embodiment, mutated cells (e.g., somatic or germcells, embryonic stem cells or adult multipotential stem cells) can beused to create a transgenic animal. Methods for making transgenicanimals from embryonic stem cells are well known in the art (see forexample, Jackson and Abbott (2000) Mouse Genetics and Transgenics,Oxford University Press, pgs. 266-284; and Hogan, Beddington, Costantim,and Lacy (1994) Manipulating the Mouse Embryo, Cold Spring Harbor Press,all pages; Joyner, Bioessays 13:649-656 (1991); each referenceincorporated herein by reference for teaching methods of producingtransgenic animals from stem cells). Similarly, methods for makingtransgenic animals from somatic or germ cells are well known in the art(see, e.g., U.S. Patent Nos. 5,322,775, 5,366,894, 5,476,995, 5,650,503and 5,861,299, all of which are incorporated herein by reference intheir entireties for teaching methods of producing transgenic animalsfrom mutated or genetically manipulated somatic or germ cells). One suchmethod is nuclear transfer cloning, in which the nucleus of a donorsomatic cell is genetically modified (e.g., using the mutational methodsof the present invention), and then the nucleus is removed from thedonor cell and placed into a recipient cell (preferably, an oocyte) toproduce a transgenic animal containing the genetic modifications fromthe donor nucleus. This process is well-known in the art and will befamiliar to the ordinarily skilled artisan (see, e.g., Campbell et al.,Nature 380:64-66 (1996); Cibelli et al., Nature Biotech. 16:620-621(1998); McCreath et al., Nature 405:1066-1069 (2000); Hochedlinger etal., Nature 415:1035-1038 (2002); Schnieke et al., Science 278:2130-2133(1997); Kasinathan et al., Nature Biotech. 19:1176-1178 (2001); Wolf etal., Arch. Med. Res. 32:609-613 (2001); the disclosures of all of whichare incorporated herein by reference in their entireties).

[0193] In one embodiment, transgenic animals that contain an insertionalmutagen that is associated with a specific mutated gene can be preparedby, for example, physicochemically or insertionally mutating sperm cellsin vivo and physicochemically or insertionally mutating oocytes in vitroor in vivo (e.g., one or more lentiviral vectors), and then fertilizingthe mutated oocytes with the mutated sperm cells to produce a homozygousmutant zygote. This zygote can then be implanted into a recipient femaleand carried to term, thereby producing a transgenic animal homozygousfor one or more mutations. Other methods for producing transgenicanimals are well-known in the art, and will be familiar to theordinarily skilled artisan (see, e.g., WO 90/05188; Hammer, R.E., etal., J. Animal Sci. 63:269-278 (1986); Pursel, V.G., et al., J. Reprod.Fert. Suppl. 40:235-245 (1995); Houdebine, L.J. Biotechnol. 34:269-287(1994); Hammer, R.E., et al., Nature 315:680-683 (1985); Mortensen,R.M., et al., Mol. Cell. Biol. 13:2391-2395 (1992); Deng, C., et al. ,Cell 82:675-684 (1995); and Murakami, T., et al., Devel. Gen. 10:393-401(1989), the disclosures of all of which are incorporated herein byreference in their entireties).

[0194] Transgenic animals can be created in any eukaryotic organism. Inpreferred embodiments, the transgenic organism is a fly, a worm, a fish,or a mammal. In highly preferred embodiments, the organism is a human, anon-human primate, a mouse, a rat, a pig, a cow, a sheep, a dog, a cat,a bird, a zebrafish, C. elegans, or Drosophila. The transgenic animalcan be used to carry out genetic screens for phenotypes of interest orfor studying the function of individual genes. Examples of phenotypesinclude, but are not limited to, weight, height/length, organ histology,organ function, immune competency, blood chemistry (e.g., cholesterollevels, etc.), bone density and structure, gross morphology, andbehavior. Additional phenotypic screens are known in the art and usefulin the present invention (see for example Nolan et al., Nature Genetics25:440-443 (2000); incorporated herein by reference for teachingphenotypic screens).

[0195] In another embodiment, multicellular organisms can be mutateddirectly by in vivo mutagenesis. An animal can be produced from a cellthat is mutated in vitro. Mutagenesis can be physicochemical orinsertional. The cell can be a stem cell (embryonic or adult), somaticcell, or germ cell. The mutation that is introduced into the animal thisway could be a heterozygous mutation in a gene where a homozygousmutation is necessary to produce a phenotype in an organism or in a cellin an organism. The organism can be mutagenized directly to produce thehomozygous mutation. A gene responsible for the phenotype can beidentified by a tag in the cell used to make the animal or in a cellinsertionally mutagenized in the intact animal. The mutation that isintroduced may be part of a set of mutations (i.e., mutation in two ormore different genes) that are all required to produce a phenotype in anorganism or in a cell in an organism. The organism can be mutagenizeddirectly to produce the other required mutations.

[0196] Alternatively, transgenic plants can also be produced accordingto the methods of the present invention. In such methods, one or more,or suitably two or more, genes or alleles in a plant cell is mutatedaccording to the methods of the invention. Transgenic plants may then beprepared using this mutated genomic DNA according to art-known plantgenetic engineering techniques, such as nuclear transfer, transformationor protoplast fusion (see Hall, Robert D., Plant Cell Culture Protocols,Humana Press, New Jersey (1999); Gartland and Davey, AgrobacteriumProtocols, Humana Press, New Jersey (1995); Kosuge et al., Gen. Eng. ofPlants 26:5(1983); Rogers et al., in: Methods for Plant MolecularBiology, A. Weissbach and H.eds., Academic Press, Inc., San Diego, CA(1988)). Such techniques are widely in use (see, e.g., Chaleff, R.S.,Genetics of Higher Plants: Applications of Cell Culture, Cambridge:Cambridge University Press (1981)), and newly inserted foreign geneshave been shown to be stably maintained during plant regeneration andare transmitted to progeny as typical Mendelian traits (Horsch et al.,Science 223:496 (1984), and DeBlock et al., EMBO 3:1681 (1984)). Theseforeign genes retain their normal tissue specific and developmentalexpression patterns. The Agrobacterium tumefaciens-mediatedtransformation system has also proved to be efficient for transfer ofgenetic material, including many dicotyledonous plant species (Barton etal., Cell 32:1033(1983); Chang et al., Planta:551-558 (1994)) andmonocotyledonous plants, e.g., in plants in the Liliaceae andAmaryllidaceae families (Hooykaas-Van Slogteren et al., Nature311:763-764 (1984)) and in Dioscorea bulbifera (yam) (Schafer et al.,Nature 327:529-532 (1987)).

[0197]Identification of conditional mutationsConditional mutations thatproduce phenotypes only after imposing specific experimentallycontrolled conditions can also be generated using the proceduresdescribed in this application. These conditional mutations can be usefulin enabling more detailed investigation of gene function and access to awider range of phenotypes that become possible as a consequence of theinherent ability to precisely control the timing and degree of functionof conditionally mutant gene products. Examples of conditional mutationsinclude the creation of cold or heat sensitive mutant cells or organismsthat exhibit the loss of mutant gene function and the consequentappearance of mutant phenotypes only under conditions of depressed orelevated temperature, respectively (references 1-6), or the creation ofchemically destabilized alleles that depend upon, for example, DMSOexposure, to uncover the altered function of the mutant alleles(reference 7).

[0198] The examples described above identify alleles that areconditional upon changes in the environment of the cell or organism.Other alleles that are conditional upon changes that are intrinsic tothe mutated cell can also be identified. In this embodiment,experimentally controlled changes in the activity of components of thecell or organism would alter the function of conditionally mutant geneproducts, and this regulation of mutant gene function would serve toalso regulate the appearance of the mutant phenotype. For example, Hsp90and other chaperonin proteins have been shown to be required to maintainthe active conformation of many marginally stable proteins includingproteins that contain destabilizing sequence changes as a result ofmutation (reference 8-11). Engineering cells to express Hsp90 only underthe regulation of an inducible promoter (for example by usingtetracycline, ecdysone, or other inducible promoter systems to controlHsp90 gene expression), or treating cells with chemicals that abbrogateHsp90 function would generate cells in which many mutant proteins couldbe destabilized and their loss of function phenotypes revealed byexperimentally controlled manipulation of Hsp90 activity (references8-11). Thus the expression of mutant phenotypes would become dependentupon the experimentally induced reduction in Hsp90 or other chaperoninactivity. Hsp90 activity might also be manipulated, for example, bycreating cells that express their only Hsp90 protein as a fusion proteinconsisting of a steroid hormone binding (or other regulatory) domainfused to Hsp90 protein. Such a regulatory domain-chaperonin fusionprotein would only exhibit chaperone active when bound with theappropriate steroid hormone (or other regulatory ligand; references12-15) and conditional mutations and phenotypes would depend upon theconcentration of the regulatory ligand in these cells. 1: Tasaka SE,Suzuki DT. Genetics. 1973 Jul;74(3):509-20; 2: Suzuki DT. Science. 1970Nov 13;170(959):695-706; 3: Suzuki DT, Piternick LK, Hayashi S, TarasoffM, Baillie D, Erasmus U. Proc Natl Acad Sci U S A. 1967Apr;57(4):907-12; 4: Pringle JR. Methods Cell Biol. 1975;12:233-72; 5:Basilico C. Adv Cancer Res. 1977;24:223-66; 6: Meiss HK, Talavera A,Nishimoto T. Somatic Cell Genet. 1978 Jan;4(1):125-30; 7: Poloni D,Simanis V. FEBS Lett. 2002 Jan 30;511(1-3):85-9; 8: Morimoto RI, KlineMP, Bimston DN, Cotto JJ. Biochem. 1997 32: 17-29; 9: Jakob U, Lilie H,Meyer I, Buchner J. J. Biol. Chem. 1995 270:7288-94; 10: Rutherford SL,Lindquist S. Nature. 1998 Nov 26;396(6709):336-42; 11: Queitsch C,Sangster TA, Lindquist S. Nature. 2002 Jun 6;417(6889):618-24; 12:Angrand PO, Woodroofe CP, Buchholz F, Stewart AF. Nucleic Acids Res.1998 Jul 1;26(13):3263-9; 13: Tada M, O'Reilly MA, Smith JC.Development. 1997 Jun;124(11):2225-34; 14: Takebayashi H, Oida H,Fujisawa K, Yamaguchi M, Hikida T, Fukumoto M, Narumiya S, Kakizuka A.Cancer Res. 1996 Sep 15;56(18):4164-70; 15: Metzger D, Clifford J, ChibaH, Chambon P. Proc Natl Acad Sci U S A. 1995 Jul 18;92(15):6991-5.

[0199] It will be understood by one of ordinary skill in the relevantarts that other suitable modifications and adaptations to the methods,compositions and applications described herein are readily apparent fromthe description of the invention contained herein in view of informationknown to the ordinarily skilled artisan, and can be made withoutdeparting from the scope of the invention or any embodiment thereof.Having now described the present invention in detail, the same will bemore clearly understood by reference to the following examples, whichare included herewith for purposes of illustration only and are notintended to be limiting of the invention.

[0200]EXAMPLES EXAMPLE 1:Combined Mutagenesis of Embryonic StemCellsEmbryonic stem cells (RI) (Nagy et al., Proc. Natl. Acad. Sci. USA90:8424-8428 (1990)) are cultured on feeder cells or gelatin coatedplates at 37EC in a humidified incubator with 7.5% CO₂. The cells arecultured in D-MEM/15% FBS/0.1 mM β-mercaptoethanol/Leukemia InhibitoryFactor (1000 U/ml)/non-essential amino acids. The cells are grown to 80%confluence. In 10 plates, each containing approximately 4 x 10⁶ cells,O⁶-benzylguanine (O⁶-BG) is diluted in DMSO and added to the media to 10µM final concentration. To a separate 10 plates, no O⁶-BG is added.Following a 12-16 hour incubation, cells from each plates aretrypsinized, diluted to 5 x 10⁶ cells/ml, and incubated with variousconcentrations of ENU for 1 or 2 hours. ENU concentrations were 0.2mg/ml, 0.3 mg/ml, 0.35 mg/ml, and 0.4 mg/ml. The cells that arepretreated with O⁶-BG were incubated with O⁶-BG during the ENUtreatment. Following treatment, cells from each ENU treatment arewashed, trypsinized to dissociate aggregates, counted, and plated induplicate into gelatin coated tissue culture plates at between 100,1000, 10,000, and 100,000, cells per plate and grown under standardconditions described above. The O⁶-BG treated cells are grown in thepresence of O⁶-BG (10 µM) for 24 hours, followed by growth in standardmedia. Colonies are counted after 10 days. In the duplicate plates,6-thioguanine (6-TG) is added at 48 hours post-plating to select forHPRT-null mutations in order to determine the mutation frequencyproduced by each treatment condition. The number of 6-TG resistantcolonies divided by the number of colonies present on the duplicate(non-6TG selected) plate determines the mutation frequency for a singlecopy gene.

[0201] In an example of the methods of the invention, a plate containingapproximately 1000 clones is selected from an ENU/O⁶-BG treatmentcondition that give a mutation frequency of 1 in 1000. The cells areexpanded to 5 x 10⁹ cells (50% confluence) under standard cultureconditions described above. The cells are then washed and incubated withpKO-1 retroviral vector at an MOI = 1 for 24 hours. The structure ofpKO-1 is shown in Figure 3B. After 24 hours, the cells are washed andreplated in duplicate into fresh media at 10% confluence. One of theduplicate libraries is grown to 40-50% confluence and placed tinder G418selection (250 µg/ml). The other duplicate library is grown toconfluence, trypsinized, frozen in 90%FBS/10%DMSO at 10⁷ cells/ml, andstored in liquid nitrogen. Cells grown under G-418 selection are grown,without replating, for 10-12 days with several media changes. G418resistant clones are counted, trypsinized, and expanded. The library canthen be stored in liquid nitrogen or used for phenotypicscreening.EXAMPLE 2:Combined Mutagenesis of Jurkat CellsCellular screento identify gene knockouts that cause resistance to FasL-inducedapoptosis in Jurkat cellsThe combined mutagenesis technology describedin this application was developed to speed the discovery of genefunction in mammalian cell culture by coupling distinct mutagenesistechniques to efficiently generate phenotypes and simultaneously enablethe facile identification of gene mutations that cause these phenotypes.combined mutagenesis combines the strengths of various mutagenicapproaches to compensate for distinct deficiencies of each of theindividual mutagenesis techniques. The embodiment of combinedmutagenesis that is described here uses the following sequence of stepsto identify cell based gene function (illustrated in Figure 1):·highdensity ENU mutagenesis of cells in culture; creates compact librariesof cells that contain mutations in nearly all genes·select only thoseENU mutagenized clones for further gene trap mutagenesis that do notalready display the phenotype·gene trap mutagenesis of selected ENUclones·selection for gene trap mutagenized clones that now display thephenotype·test to ensure the phenotype reverts to wild type uponCre-mediated removal of the gene trap·sequence identification of theinsertionally mutated gene in Cre-revertible gene trap clones·siRNAconfirmation that specific gene knockouts are sufficient to conferphenotypeEfficient creation of phenotypes and identification of mutatedgenes derives from combining the high efficiency of chemical mutagenesiswith the ability of gene trap insertional mutagens to provide a sequencetag for identification of the mutated genes (Figure 15). Chemicalmutagens are highly efficient means to generate high densities ofmutations in mammalian cells, and the size of libraries that containmutations in all genes can be less than 50 cells. However, theidentification of the alleles that cause specific phenotypes in thesechemically mutated cell lines is extremely difficult. In contrast, whilemutagenesis by gene trap vectors is much less efficient, these gene trapmutagens compensate by enabling the easy identification of the genesthat are responsible for a given phenotype by means of the physicallinkage between the mutagenized gene and sequences in the insertionalmutagen. Cells showing phenotypes created by combined mutagenesis carryat least one chemically induced mutation and at least one gene trapmutation whose mutual interaction is needed to generate the cellularphenotype (Figure 15).

[0202] To test the combined mutagenesis technology we needed to 1)establish a high density chemical mutagenesis protocol, 2) construct aneffective and reversible gene trap retrovirus, 3) create a combinedmutagenesis mutagenized library with these reagents, and 4) conduct aproof-of-principle screen of this library to identify cells that haveacquired resistance to Fas-induced apoptosis as a result of the combinedmutagenesis. The development and testing of combined mutagenesis isdescribed below.

[0203] The ability to identify all genetic mutations that can create aphenotype requires that large numbers of mutations be created in as fewcells as possible, and the high efficiency of ENU chemical mutagenesisis suited to this task. We set out to determine the ENU treatment neededto create 50-100 cell libraries in which all genes are mutated at leastonce (defined as a 1x knockout library) in Jurkat cells. To characterizethe mutagenesis we measured both cell survival and the mutation rate incharacterizing the optimal ENU treatment (Table I). Survival wasdetermined by limiting dilution, and the mutation rate was estimated inthe population of surviving clones based on the frequency of knockingout the single copy HPRT gene (assayed by the survival of such HPRT⁻cells upon culture in the presence of 6-thioguanine). As shown in thetable below, at an ENU dose of 0.2mg/ml the mutation rate is 0.02 HPRT⁻genes/cell and the 1x library size is therefore ~50 cells. Creation oflibraries of these densely mutagenized cells provides a geneticallysensitized background in which small increments of further mutagenesiscan create phenotypes by knocking out the sole remaining wild typealleles of ENU-mutated genes.

[0204]Table I:ENU dose(mg/ml)% survivalmutation rate1x librarysize*098.40%3.9x10^(-7)2.56x10^60.11.83%2.4x10^(-3)4170.1250.93%4.9x10^(-3)2040.151.08%6.1x10^(-3)1640.1750.88%8.2x10^(-3)1220.20.29%2.0x10^(-2)500.250.07%3.3x10^(-2)310.457x10^(-5)NANA*1x library size refers to the number of ENU treated clones needed tocontain mutations. Chemical mutagenesis protocol:Jurkat cells weretreated with 10uM O-6-BG (Sigma) at 5x10^5 cells/ml for 16 hours at 37ºCin a humidified incubator with 5% CO2. The pretreated cells were thenincubated with desired concentration of ENU (Sigma) in the presence ofO-6-BG for 2 hours at 37ºC with constant shaking. At the end ofincubation cells were washed with 10-volume 1xPBS three times andresuspended in complete media with 10uM O-6-BG. 24 hours later O-6-BGwas washed off, cells were resuspended in complete media and seeded onto96-well plate at 20 cells per well or 400-2000 cells per well. Cellswere then cultured for 10 days. Viability is determined by the lowerdensity seeding based on number of wells containing growing colonies. Todetermine mutation frequency, cells seeded at the higher density wereused to select for Hprt-loss of function mutants in the presence of 40uM6-TG (Sigma). Number of wells containing growing colonies in thepresence of 6-TG was scored 7-10 days later. Mutation frequency iscalculated as # of 6-TG resistant colony divided by # of viable cellsseeded.

[0205] Similarly mutated libraries were created by treating Jurkat cellswith 0.7mg/ml EMS (Sigma) following above protocol A. 1x library sizecontained about 100 cells under this condition.

[0206] Karyotype of ENU mutagenized Jurkat cells:In some preferredembodiments, cells subjected to mutation are diploid. Therefore wedetermined the chromosome count of Jurkat cells and ENU mutagenizedJurkat cells by Giemsa stain. ~75% of ENU mutagenized Jurkat cells arediploid or hypodiploid. This is comparable with wild type Jurkat cells.

[0207] In order to prepare the ENU libraries for gene trap mutagenesis,the surviving Jurkat clones were then tested to ensure that theyremained susceptible to FasL induced apoptosis after the 0.2mg/ml ENUmutagenesis; 100 of these Fas-sensitive clones were then chosen astargets for gene trap mutagenesis. This exercise demonstrates that wecan create densely mutagenized libraries of cells that do not exhibitthe phenotype of interest even though they contain knockouts in one copyof 1-3% of the genes in each cell. This combination of dense mutagenesiswith lack of phenotype is crucial since it creates small and tractablelibraries of cells that possess genetic backgrounds in which single genetrap knockouts can create a phenotype by disrupting the remaining wildtype copy of genes that determine phenotype.

[0208] Creation of an efficient gene trap vector was needed to serve asan insertional knockout mutagen and tag of the insertionally mutatedgenes for the next phase of combined mutagenesis function discovery. Thedesign of the gene trap retroviral vector pDKO2 that was created forthis purpose is shown below (Figure 16). This vector is designed to traptranscriptional active genes using the function of the splice acceptorin the vector. When the vector is integrated within a gene, splicingoccurs from splice donors that exist at the end of exons in the trappedendogenous gene onto the splice acceptor that is provided by the vector.Once this splicing event occurs, a fusion transcript will be made thatresults in the introduction of vector encoded stop codons that cause thepremature termination of the protein product of the trapped gene.Downstream IRES (internal ribosome entry site) activity in the fusiontranscript enables the reinitiation of translation on the fusiontranscript and this translation expresses a selectable drug resistanceprotein. Expression of drug resistance allows selection to identify thetrapping of transcriptionally active genes. Lox signals flank the genetrap portion of pDKO2, and Cre-mediated recombination of these lox sitesresults in the deletion of the pDKO2 gene trap and knockout functions;this deletion should revert phenotypes that depend upon insertionalmutagenesis and aid in the identification of biologically active genetraps.

[0209] The function of the various components of pDKO2 were separatelytested to ensure activity as follows:·IRES: We confirmed that this IRESdoes function to reinitiate translation, and showed that it does nothave promoter activity in pDKO2·TK: expression of TK was shown to causecell death in gancyclovir containing media·Cre/lox: transfection of creexpression plasmids into cells carrying the integrated pDKO2 constructresulted in excision events occurring in ~80% ofcells·S/A--x--IRES--DR--bGHpA: the gene trap portion of pDKO2 wasintroduced into Jurkat cells, followed by selection for drug resistance(neomycin 1.5mg/ml for Jurkat), drug resistant clones were harvested andthe appearance of drug resistance was associated with splicing ofendogenous transcripts onto the pDKO2 splice acceptor in all casesexamined by RT-PCRProduction of the pDKO2 retroviral vector occurred bytransfecting pDKO2 into RetroPack PT67 cells (Clontech) via Exgen500(MBI Fermentas). Individual stable colonies were picked and selected forhigh titer producers. High titer virus soup was harvested and used toinfect Jurkat cells following spin-infection protocol. Briefly, 3x106Jurkat cells were resuspended in 2ml complete media plus 1ml viral soupand polybrene at 8ug/ml. We spininfected at 1000g for 1 hour. Cells werethen placed in 32C incubator overnight and then incubated at 37C for 24hours to allow integration and expression of retrovirus. Titer wasdetermined by limiting dilution and found to be 1-3x104 per ml.

[0210] To assess gene trap efficiency of this vector, we assayed thefrequency of gene trap events for several known genes by RT-PCR. Twopairs of nested gene specific primers (specific for DHFR, HPRT, FasR,and Casp8) were used in this analysis, and the resultant RT-PCR productswere sequenced to confirm the identity of gene traps. All were confirmedto be true gene trap events with upstream exons from the endogenous genecorrectly spliced onto the splice acceptor in pDKO2. In most cases pDKO2was found to integrate within introns near the 5"-end of the trappedgene. The data shown below suggest that the 1x library size for trappinga single allele is ~2x10^4 clones. It is easy to generate this number ofclones, and this efficiency permits the use of pDKO2 to create genomewide gene trap knockout libraries in each of the Fas-sensitive ENUclones described above.

[0211]Gene trap frequency:Cell type# clones per pool# pools total#clones total# of pools in which one of the following genes istrappedHPRTDHFRFasRCasp8Jurkat7500107.5x10^42339Characterization oftrapped genes:gene nameHPRTDHFRFasRCasp8# traps2339#alleles/cell1222gene size40kb30kb26kb55kbFor 75,000clones(2+3+3+9)/(1+2+2+2)=2.4 hits per allelefor a diploid gene: 4.8hits per 75,000 clonesaverage size for assayed genes:(40+30+26+55)/4=38kb for average gene size of 28kb, 1x library size:2x10^4The next step is to combine these elements to implement a combinedmutagenesis screen to identify gene functions. Clones from the ENUJurkat library were infected with the pDKO2 gene trap retrovirus tocreate a combined mutagenesis knockout library in Jurkat cells (Figure17). One hundred ENU mutagenized Jurkat clones (2x genome coverage forENU mutagen) that had been tested to ensure unchanged sensitivity toFas-induced apoptosis were chosen for this pDKO2 retrovirus infectionand ~10^4 drug resistant clones (0.5x genome coverage for pDKO2 mutagen)were obtained for each ENU clone. The final combined mutagenesis librarytherefore consists of ~one million clones and this number represents ~1xcoverage of the range of possible human gene disruptions in these cells.This coverage is sufficient to identify the majority (but not all) ofthe mutations that decrease FasL induced apoptosis in Jurkat cells.Under above conditions, the spontaneous Fas resistance rate is ~1 in2-3x10^62.Mutation screen:ENU clones were first tested for FasAbsensitivity. Only Fas-sensitive clones were carried forward for thescreen. 100 ENU clones (derived from 0.2mg/ml ENU treatment, table A.1.)were expanded (~2x library coverage) to 5-10x106 cells per clone. EachENU clone was infected with pDKO2-neo retrovirus to create a pool ofcells where one allele of a gene can be potentially mutated by the pDKO2vector. About 104 drug resistant clones were obtained for each ENUclone, which is about 0.5x library coverage for gene trap event. Cellswere split into duplicate, A and B, immediately after infection and weresubsequently grown in neomycin containing media for 10-12 days to selectfor neomycin resistant clones and to deplete endogenous wild typeprotein produced prior to the mutation of the gene. From each pool A orB, 0.5-1x106 cells (100-200 cells per clone) were taken into Fas screenfollowing protocol in C1. Fas resistant clones were identified by theoutgrowth of the cells during the first 2-3 weeks after Fas treatment.

[0212] Fas resistant clones were subjected to re-test by Annexin Vstaining (Molecular Probes) and caspase 3 activity assay (Intergen)(Ref: Blood 84, 1415, 1994; J. Biol. Chem. 273, 32608-32613, 1998). ~70%of the clones confirm the Fas resistant phenotype based on these twoassays. To determine if the resistant phenotype is due to insertion ofthe pDKO2 vector into the genome, cre expression plasmid was transfectedinto each clone and followed by gancyclovir selection to select forexcision event. Clones were then analyzed for reversion of thephenotype, thereby demonstrating that the trapped gene was responsiblefor the phenotype.

[0213] FasL induced apoptosis is well studied in Jurkat cells, andmodulators of this pathway have potential clinical relevance in cancer,stroke and other disease processes. Previous genetic and biochemicalanalysis many systems and laboratories have established the basic signaltransduction path involved in initiating FasL induced apoptosis (Figure18). We expect to identify combined mutagenesis gene trap knockouts ofsome of the members of the known Fas signaling pathway (Figure 18) asone result of this screen. Knockout of novel genes might also revealpreviously unknown players important for regulating apoptosis, and thesenovel regulatory genes are of great interest as potential new targetsfor therapeutic intervention.

[0214] To identify gene traps that affect sensitivity to FasL, thedoubly mutagenized combined mutagenesis library was selected for clonesthat exhibited resistance to Fas-induced apoptosis. Under the FasLselection conditions, there is a low spontaneous background ofresistance that occurs at ~1 in 2-3x10⁶ cells, and we recovered 35Fas-resistant clones from the million clone library. The phenotype ofeach clone was further confirmed by showing that FasL treatment failedto activate the normal levels of Annexin V staining and Caspase 3activity as molecular markers of apoptosis. These results show thatcombined mutagenesis can efficiently create reproducible phenotypes inmammalian cells.

[0215] To determine which of the FasL-resistant cells have acquiredtheir phenotype as a direct result of the insertion of the pDKO2 vectorinto the genome, we used transfection of Cre expression plasmids toinduce the excision of pDKO2 gene trap sequences from each of theclones. Cre mediated recombination was followed by gancyclovir selectionto detect deletion of the thymidine kinase element along with the genetrap functions in pDKO2. Clones were then analyzed for reversion of thephenotype, and 13 out of the 35 clones showed phenotypic reversion afterCre-mediated removal of the gene trap (phenotypic reversion data shownin Figure 19). The cells that did not show phenotypic reversion afterexcision of the gene trap may have acquired resistance to FasL as theresult of a spontaneous mutation, but since such events are nearlyimpossible to identify, these clones were not analyzed further. Thirteenclones did show FasL resistance that depended upon gene trap knockout,and these cells were further studied to identify the trapped genes andto characterize their role in apoptosis.

[0216] The 13 clones showing FasL resistance caused by the pDKO2 genetrap were analyzed using RT-PCR, 5"-RACE and inverse PCR to identify thebiologically active trapped genes. It has been demonstrated previouslythat mutation of caspase-8 gene leads to FasL resistance (Curr. Biol.8(18), 1001-1008, 1998). To test whether caspase-8 has been mutated inany of the FasL resistant clones from our screen, we carried out RT-PCRassay with two nested caspase-8 specific forward primers and two nestedvector specific reverse primers on those clones. We identified caspase-8trap in 2 of the clones. 5"-RACE and inverse PCR identified the trappedgenes in most of the other Fas-resistant clones. Many of these genes hadnot been previously characterized as having an involvement in FasLinduced apoptosis, and these genes are expected to play unexpected rolesin apoptosis and as potential therapeutic targets.

[0217] In summary, we have developed a loss of function geneticsstrategy that enables the discovery of in vivo gene function inmammalian cells, and have shown that this combined mutagenesis strategypermits the rapid identification of known and novel genes that performthese functions. Such a genetic approach should be valuable in theidentification of gene function and in the discovery of new targets fortherapeutic intervention.

[0218] Having now fully described the present invention in some detailby way of illustration and example for purposes of clarity ofunderstanding, it will be obvious to one of ordinary skill in the artthat the same can be performed by modifying or changing the inventionwithin a wide and equivalent range of conditions, formulations and otherparameters without affecting the scope of the invention or any specificembodiment thereof, and that such modifications or changes are intendedto be encompassed within the scope of the appended claims.

[0219] All publications, patents and patent applications mentioned inthis specification are indicative of the level of skill of those skilledin the art to which this invention pertains, and are herein incorporatedby reference to the same extent as if each individual publication,patent or patent application was specifically and individually indicatedto be incorporated by reference.

Claims 1.A cell library comprising 1-10⁶ physicochemically mutatedclones wherein one of more of said physicochemically mutagenized cloneshas been subjected to insertional mutagenesis. 2.A cell librarycomprising 1-10⁵ physicochemically mutated clones wherein one of more ofsaid physicochemically mutagenized clones has been subjected toinsertional mutagenesis. 3.A cell library comprising 1-10³physicochemically mutated clones wherein one of more of saidphysicochemically mutagenized clones has been subjected to insertionalmutagenesis. 4.A cell library comprising 50-100 physicochemicallymutated clones wherein one of more of said physicochemically mutagenizedclones has been subjected to insertional mutagenesis. 5.A cell librarycomprising about 30-about 400 physicochemically mutated clones whereinone of more of said physicochemically mutagenized clones has beensubjected to insertional mutagenesis. 6.A cell library comprising 2, 5,10, 25, 50, 100, 200, 400, 600 or 800 physicochemically mutated cloneswherein one of more of said physicochemically mutagenized clones hasbeen subjected to insertional mutagenesis. 7.The cell library of any ofclaims 1-6 wherein total coverage of physicochemically mutated allelesin the genome is 0.1X-10X. 8.The cell library of claim 7 wherein totalcoverage of physicochemically mutated alleles in the genome is 0.1X,1.0X, 5X or 10X. 9.The cell library of any of claims 1-6 wherein theaverage number of physicochemically mutated alleles per cell is 10-100,250-5,000, 50-10,000 or 10,000-50,000. 10.A method for creating amutagenized cell library, said method comprising insertionallymutagenizing a cell library comprising 1-10⁶ physicochemically mutatedclones. 11.A method for creating a mutagenized cell library, said methodcomprising insertionally mutagenizing a cell library comprising 1-10⁵physicochemically mutated clones. 12.A method for creating a mutagenizedcell library, said method comprising insertionally mutagenizing a celllibrary comprising 1-10³ physicochemically mutated clones. 13.A methodfor creating a mutagenized cell library, said method comprisinginsertionally mutagenizing a cell library comprising 50-100physicochemically mutated clones.
 14. 14A method for creating amutagenized cell library, said method comprising insertionallymutagenizing a cell library comprising about 30-about 400physicochemically mutated clones. 15.A method for creating a mutagenizedcell library, said method comprising insertionally mutagenizing a celllibrary comprising 2, 5, 10, 25, 50, 100, 200, 400, 600 or 800physicochemically mutated clones.
 16. The method of any of claims 10-15wherein in the cell library total coverage of physicochemically mutatedalleles in the genome is 0.1X-10X.
 17. The method of any of claims 10-15wherein in the cell library total coverage of physicochemically mutatedalleles in the genome is 0.1X, 1.0X, 5X or 10X. 18.The method of any ofclaims 10-15 wherein in the cell library the average number ofphysicochemically mutated alleles per cell is 10-100, 250-5,000,50-10,000 or 10,000-50,000.